Sandra Imbrogno

β3‐Adrenoceptors modulate left ventricular relaxation in the rat heart via the NO‐cGMP‐PKG pathway

Aims:  Using a model of isolated and Langendorff‐perfused rat heart we analysed whether activation of β3‐adrenergic receptors (β3‐ARs) influences ventricular lusitropic performance. We also focused on the NOS/NO/cGMP/PKG cascade as the signal transduction mechanism.

Beta3-adrenoceptor in the eel (Anguilla anguilla) heart: negative inotropy and NO-cGMP-dependent mechanism.

SUMMARY Neuroendocrine regulation of cardiac function involves a population of three types of β-adrenoceptors (ARs). In various mammalian species,β 1- and β2-AR stimulation produces an increase in contractility; whereas β3-AR activation mediates negative inotropic effects. At the moment, nothing is known about the physiological role of β3-AR in fish. Using an isolated working heart preparation, we show that a β3-AR selective agonist BRL37344 (0.1-100 nmol l-1) elicits a dose-dependent negative inotropism in the freshwater eel Anguilla anguilla. This effect was insensitive to the β1/β2-AR inhibitor nadolol (10 μmol l-1), but was blocked by theβ 3-AR-specific antagonist SR59230 (10 nmol l-1). The analysis of the percentage of stroke work (SW) variations, in terms of EC50 values, induced by BRL37344 alone (10 nmol l-1), and in presence of SR59230 (10 nmol l-1), indicated a competitive antagonism of SR59230. In addition to the classic positive inotropism, the non-specific β agonist isoproterenol (100 nmol l-1) induced, in 30% of the preparations, a negative inotropic effect that was abrogated by pre-treatment with SR59230, pointing to a β3-mediated pathway. The BRL37344-induced negative inotropic effect was abolished by exposure to a Gi/o proteins inhibitor pertussis toxin (PTx; 0.01 nmol l-1), suggesting a Gi/o-dependent mechanism. Using L-N5(l-imino-ethyl)ornithine (L-NIO; 10 μmol l-1), as a nitric oxide (NO) synthase (NOS) blocker and haemoglobin (Hb; 1 μmol l-1), as a NO scavenger, we demonstrated that NO signalling is involved in the BRL37344-induced response. Pre-treatment with either an inhibitor of soluble guanylate cyclase (GC) 1H-(1,2,4) oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ; 10 μmol l-1), or an inhibitor of the cGMP-activated protein kinase (PKG) KT5823 (100 nmol l-1), abolished the β3-dependent negative inotropism, indicating the cGMP-PKG component as a crucial target of NO signalling. Taken together, our findings provide functional evidence for the presence ofβ 3-like adrenoceptors in the eel Anguilla anguilla heart identifying, for the first time in a working fish heart, theβ 3-AR-dependent negative inotropy discovered in mammals.

Angiotensin II-induced inotropism requires an endocardial endothelium-nitric oxide mechanism in the in-vitro heart of Anguilla anguilla.

SUMMARY Using an isolated working heart preparation we show that angiotensin II (ANG II), at concentrations of 10-10–10-7 mol l-1, elicits negative chronotropism and inotropism in the freshwater eel Anguilla anguilla. The negative inotropism was insensitive to losartan and CGP42112 (AT1 and AT2 ANG II receptor antagonists, respectively), and was abrogated by the AT1 receptor antagonist CV11974, the G protein blocker pertussis toxin (PTx) and the muscarinic antagonist atropine. In contrast, it was not affected by the adrenoceptor antagonists propanolol, sotalol and phentolamine. Using donors (l-arginine) and inhibitors [NG-monomethyl-L-arginine (l-NMMA), l-N5(1-iminoethyl)ornithine (L-NIO)] of nitric oxide synthase (NOS), and haemoglobin as NO scavenger, we demonstrate that NO signalling is involved in ANG II-mediated inotropism. Pretreatment with Triton X-100, a detergent that damages the endocardial endothelium (EE), or with 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase, or with the cGMP-activated protein kinase (PKG) inhibitor KT5328, abolished ANG II-mediated inotropism. Thus, ANG II-mediated inotropism occurs via an EE-NO-cGMP-PKG mechanism. ANG II did not affect the mechanical performance influenced by preload changes (i.e. the Frank–Starling response), which in the eel heart is modulated by NO. This EE-paracrine-mediated cardio-suppressive action of endoluminal ANG II suggests that the hormone plays an important intracardiac role in the fish heart.

Phospholamban and cardiac function: a comparative perspective in vertebrates

Phospholamban (PLN) is a small phosphoprotein closely associated with the cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLN tonically inhibits the SR Ca‐ATPase (SERCA2a), while phosphorylation at Ser16 by PKA and Thr17 by Ca2+/calmodulin‐dependent protein kinase (CaMKII) relieves the inhibition, and this increases SR Ca2+ uptake. For this reason, PLN is one of the major determinants of cardiac contractility and relaxation. In this review, we attempted to highlight the functional significance of PLN in vertebrate cardiac physiology. We will refer to the huge literature on mammals in order to describe the molecular characteristics of this protein, its interaction with SERCA2a and its role in the regulation of the mechanic and the electric performance of the heart under basal conditions, in the presence of chemical and physical stresses, such as β‐adrenergic stimulation, response to stretch, force–frequency relationship and intracellular acidosis. Our aim is to provide the basis to discuss the role of PLN also on the cardiac function of nonmammalian vertebrates, because so far this aspect has been almost neglected. Accordingly, when possible, the literature on PLN will be analysed taking into account the nonuniform cardiac structural and functional characteristics encountered in ectothermic vertebrates, such as the peculiar and variable organization of the SR, the large spectrum of response to stresses and the disaptive absence of crucial proteins (i.e. haemoglobinless and myoglobinless species).

The catecholamine release-inhibitory peptide catestatin (chromogranin A344-364) modulates myocardial function in fish

SUMMARY Catestatin (CST), the 21-amino acid, cationic and hydrophobic peptide proteolytically derived from the ubiquitous chromogranin A (CgA), is an endogenous inhibitor of catecholamine release, a potent vasodilator in vivo and an anti-hypertensive agent in mammals, including humans. Recently, we discovered that CST also functions as an important negative modulator of heart performance in frog and rat. To gain an evolutionary perspective on CST cardiotropism in fish, we analysed the influence of bovine CST (CgA344-364) on the eel heart, as well as the eventual species-specific mechanisms of its myocardial action. Experiments were carried out on fresh-water eels (Anguilla anguilla L.) using an electrically paced isolated working heart preparation. Stroke volume and stroke work were used as measures of ventricular performance. Under basal conditions, CST (from 11 nmol l–1 to 165 nmol l–1) caused a concentration-dependent negative inotropism, which was abolished by inhibitors of either β1/β2 (propranolol) or β3 (SR59230) adrenergic receptors, or by Gi/o protein (PTx) or nitric oxide synthase (L-NMMA), or guanylate cyclase (ODQ) blockers. This suggests a β-adrenergic receptor-Gi/o protein-NO-cGMP-dependent mechanism. By contrast, the CST-induced cardio-suppression was not influenced by atropine, unspecific muscarinic antagonist, thus excluding cholinergic receptor involvement. CST also counteracted the adrenergic (isoproterenol)-mediated positive inotropism. Under increased preload (i.e. Frank–Starling response) conditions, CST induced a significant increase of the Frank–Starling response, which was blocked by L-NMMA and thapsigargin, but independent from guanylate cyclase. In conclusion, this is the first report in fish that CST modulates myocardial performance under basal, as well as under increased preload, conditions and counteracts the adrenergic-mediated positive inotropism, which strikingly supports the evolutionary significance and establishes the cardioactive role of this peptide.

Cardiac morphodynamic remodelling in the growing eel (Anguilla anguilla L.)

SUMMARY The morphodynamic changes occurring during growth were evaluated in the eel (Anguilla anguilla L.) heart. Using an in vitro working heart preparation, cardiac performance of small (body mass 96.76±27.49 g; mean ± s.d.) and large (body mass 656±12 g; mean± s.d.) eels was compared under basal conditions and under loading (i.e. preload and afterload) challenges. A parallel morphometric evaluation of the ventricle was made using light and transmission electron microscope images. The small eel hearts show a basal cardiac output lower than their large counterparts (heart rate fh, 38.93±2.82 and 52.7±1.8 beats min–1, respectively; stroke volume Vs, 0.27±0.017 and 0.37±0.016 ml kg–1, respectively; means ± s.e.m.). The two groups show similar responses at increasing preload, but differ remarkably at increasing afterload. Small eel hearts decreased Vs at afterload greater than 3 kPa, in contrast to larger hearts, which maintained constant Vs up to 6 kPa. These changes in mechanical performance are related to structural differences. Compared with the small eels, the large eels show an increase in the compacta thickness and in the diameter of the trabeculae in the spongiosa, together with reduction of the lacunary spaces. The increased compacta thickness is attained by enlargements of both the muscular and vascular compartments and reduction of the interstitium; consequently, this layer appears more compacted. Both compacta and spongiosa show higher number of myocytes together with reduced cross-sectional area and myofibrillar compartment. The compacta also shows an increased mitochondrial compartment. Our results document a cardiac morphodynamic remodelling in the growing eel.

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.

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

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

The interplay between chromogranin A-derived peptides and cardiac natriuretic peptides in cardioprotection against catecholamine-evoked stress

Chromogranin A (CgA) is the major soluble protein co-stored and co-released with catecholamines (CAs) from secretory vesicles in the adrenal medulla chromaffin cells. Present in the diffuse neuroendocrine system, it has also been detected in rat and human cardiac secretory granules where it co-stores with natriuretic peptide hormones (NPs). Mounting evidence shows that CgA is a marker of cardiovascular dysfunctions (essential hypertension, hypertrophic and dilatative cardiomyopathy, heart failure) and precursor of the cardioactive peptides vasostatin-1 (VS-1) and catestatin (Cts). This review focuses on recent knowledge regarding the myocardial, coronary and anti-adrenergic actions of VS-1. In particular, the negative inotropism, lusitropism and coronary dilation effects of rat CgA1-64 (rCgA) and human recombinant STACgA1-78 (hrSTACgA1-78) are summarized with attention on their counteracting isoproterenol- and endothelin-1-induced positive inotropism, as well as ET-1-dependent coronary constriction. The interactions between vasostatins (VSs), NPs and CA receptors are proposed as a paradigm of the heart capacity to organize complex connection-integration processes for maintaining homeostasis under intense cardio-excitatory stimuli (myocardial stress).

Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts.

Vasostatins (VSs), i.e. the main biologically active peptides generated by the proteolytic processing of chromogranin A (CGA) N-terminus, exert negative inotropism in vertebrate hearts. Here, using isolated working eel (Anguilla anguilla) and frog (Rana esculenta) heart preparations, we have studied the role of the cytoskeleton in the VSs-mediated inotropic response. In both eel and frog hearts, VSs-mediated-negative inotropy was abolished by treatment with inhibitors of cytoskeleton reorganization, such as cytochalasin-D (eel: 10 nM; frog: 1 nM), an inhibitor of actin polymerisation, wortmannin (0.01 nM), an inhibitor of PI3-kinase (PI3-K)/protein kinase B (Akt) signal-transduction cascade, butanedione 2-monoxime (BDM) (eel: 100 nM; frog: 10 nM), an antagonist of myosin ATPase, and N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide (W7) (eel: 100 nM; frog: 1 nM), a calcium-calmodulin antagonist. These results demonstrate that changes in cytoskeletal dynamics play a crucial role in the negative inotropic influence of VSs on eel and frog hearts.