Introduction
The eels (bony fish in the genus Anguilla) include approximately 18 species that, for their peculiar physiological features, have become particularly interesting experimental animals. Eels have the ability to survive in many diverse, and often harsh, environments (Tesch, 2003) and to face the environmental challenges imposed by their complex life history. For example, although eels are relatively inefficient swimmers (Bone et al., 1995), following sexual maturation they face spectacular long-distance migrations (5000–6000km) from the coasts of Europe to their spawning grounds in the Sargasso Sea (e.g. Hyde et al., 1987). Such high levels of locomotor endurance require relevant metabolic and cardio-respiratory capacities (van Ginneken and van den Thillart, 2000; Ellerby et al., 2001). Indeed, their respiratory and cardio-circulatory capacity to satisfy tissue demands is related to their great ability to regulate cardiac aerobic metabolism during hypoxia (McKenzie et al., 2003). Moreover, in response to stressful stimuli, eels do not exhibit increased plasma catecholamines (Perry and Reid, 1992) or cortisol, an indicator of sublethal chronic stress (Wendelaar Bonga, 1997). Thus, the members of the genus Anguillaare considered stress-tolerant animals (McKenzie et al., 2003). In addition, as first documented by Steffensen and Lomholt (Steffensen and Lomholt, 1990) in European eel farms, this fish exhibits exceptional tolerance to very severe chronic hypercapnia (Larsen and Jensen, 1997), which implicates an elevated capacity to accumulate plasma HCO3–(Farrell and Lutz, 1975; McKenzie et al., 2002).
Of note, during migration the eel experiences hydrostatic pressure
challenges of at least 2000–3000kPa (200–300m depth) (Aoyama et al., 1999; McCleave and Arnold, 1999). It has been experimentally observed that a short exposure to high hydrostatic pressure (over 10,000kPa) does not induce significant alterations in eel aerobic metabolism (for a review, see Sébert, 2002). This has been correlated with a low sensitivity of mitochondrial red myotomal muscle to high hydrostatic pressure, evaluated in terms of oxidative phosphorylation (Sébert and Theron, 2001), and with an efficient anaerobic metabolism. Conceivably, this might balance the deleterious effects observed during the first hours of pressure exposure (Sébert, 1997).
On the basis of these premises, the eel cardiovascular system is expected to respond with a remarkable multilevel morpho-functional plasticity and adaptability to a variety of intrinsic (body size and shape, fiber type distribution in the myotomal muscle, mode of swimming, etc.) and environmental (temperature, partial pressure of oxygen, pH, environmental pollutants, etc.) factors associated with the mode of animal life (Peyraud-Waitzenegger and Soulier, 1989; Sancho et al., 2000; Bailey et al., 2000; McKenzie et al., 2003).
After a brief outline on the basic morphological design of the eel heart, this review will summarize the present knowledge regarding the regulatory mechanisms that fine-tune eel cardiac performance, including autocrine–paracrine modulation, nitric oxide synthase (NOS)/nitric oxide (NO) signaling, and the influences of factors such as temperature, exercise, hypoxia and hypercapnia.
The adult heart configuration is characterized by a remarkable morpho-functional heterogeneity (or non-uniformity) encompassing Summary
The remarkable functional homogeneity of the heart as an organ requires a well-coordinated myocardial heterogeneity. An example is represented by the selective sensitivity of the different cardiac cells to physical (i.e. shear stress and/or stretch) or chemical stimuli (e.g. catecholamines, angiotensin II, natriuretic peptides, etc.), and the cell-specific synthesis and release of these substances. The biological significance of the cardiac heterogeneity has recently received great attention in attempts to dissect the complexity of the mechanisms that control the cardiac form and function. A useful approach in this regard is to identify natural models of cardiac plasticity. Among fishes, eels (genus Anguilla), for their adaptive and acclimatory abilities, represent a group of animals so far largely used to explore the structural and ultrastructural myoarchitecture organization, as well as the complex molecular networks involved in the modulation of the heart function, such as those converting environmental signals into physiological responses. However, an overview on the existing current knowledge of eel cardiac form and function is not yet available. In this context, this review will illustrate major features of eel cardiac organization and pumping performance. Aspects of autocrine–paracrine modulation and the influence of factors such as body growth, exercise, hypoxia and temperature will highlight the power of the eel heart as an experimental model useful to decipher how the cardiac morpho-functional heterogeneities may support the uniformity of the whole-organ mechanics.
Key words: autocrine/paracrine regulation, contractility and relaxation, Frank–Starling response, neuro-humoral modulation, sarcoplasmic reticulum, transduction cascades.
Received 5 April 2013; Accepted 3 June 2013 The Journal of Experimental Biology 216, 3575-3586 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.089292
REVIEW
The eel heart: multilevel insights into functional organ plasticity
Sandra Imbrogno
all levels of cardiac organization from gross morphology to molecular biology (Katz and Katz, 1989). As illustrated by Olson (Olson, 2006), this results from the modular morphogenesis of the heart driven by distinct transcriptional regulatory programs. Hopefully, this review may also help to illustrate how the fish heart, like all vertebrate hearts, accomplishes a multilevel physiological integration, i.e. homogeneity of function, compensating for its morpho-functional heterogeneity.
I also wish to emphasize that, due to their commercial value and laboratory amenability, many eel species are now considered critically endangered, thereby calling for ethical considerations. This would imply a critical decision when choosing this animal as an experimental model for biomedical research.
Cardiac morphological design
The eel heart consists of four chambers placed in series: a sinus venosus, a thin-walled atrium, a more muscular ventricle and an outflow tract (bulbus arteriosus) (Fig.1). The peripheral venous blood flows in sequence from the sinus venosus to the atrium, to the ventricle and to the bulbus arteriosus, from where it is pumped to the gills to be oxygenated and then distributed to the body, reflowing to the heart. The sinus venosus is a large chamber separated from the atrium by the sinus valves (Yamauchi, 1980). Its structure varies between species, being formed by variable amounts of muscle and connective tissue. In Anguilla anguilla, it is mostly made up of myocardium (Yamauchi, 1980; Farrell and Jones, 1992). The single-chambered eel atrium is formed by a rim of myocardium enveloping a complex system of thin trabeculae. The inner part of the ventricle is made up of projecting cones of myocardial muscle (trabeculae) that give the ventricular wall a spongy appearance (spongiosa). The spongiosa is surrounded by an outer layer of orderly and densely arranged myocardial bundles, named compacta, perfused by an arterial supply (Tota et al., 1983). The spongiosa is avascular and the numerous trabeculae, lined by the endocardial endothelium (EE), separate the ventricular lumen into an extensive network of lacunae of different size, supplied by the intertrabecular lacunary system. Of note, the EE synthesizes and releases various autocrine–paracrine substances and holds a variety of receptors, thus acting as an autocrine–paracrine and sensor-integrator device (e.g. via EE-NOS/NO-mediated signals), as detailed below. Therefore, because of its strategic location between the endoluminal blood and the subjacent myocardium, the extensive EE surface area, typical of the teleost hearts (Tota et al., 2010; Imbrogno et al., 2011; Garofalo et al., 2012), is well designed to function as a sensor and transducer able to integrate various intracavitary physical and chemical stimuli into signaling pathways, thereby regulating the subjacent working myocardium (Imbrogno et al., 2011; and references therein). Icardo (Icardo, 2012) recently reported, in the common eel (A. anguilla), the presence of a large collagen network, extending between the subepicardium and the subendocardium, which mimics the distribution of both the myocardial bundles in the compacta and the trabeculae in the spongiosa. In particular, the subendocardial localization of the collagen in the trabeculae points to an important mechanical role in the maintenance of ventricular structure and dynamics. Noteworthy, to prevent ventricular dyssynergy between the compacta and the spongiosa, a functional synchronism is necessary. Although studies in the eel heart ventricle have not yet detailed how such synergy is attained in order to provide homogeneity of the contractile performance, desmosome-like and fascia-adherens-like structures, observed in various teleost species, could function as anchorage structures between the compacta and
the spongiosa, concurring to provide a morpho-functional linkage between the two muscular layers (Pieperhoff et al., 2009). Nervous and humoral mechanisms may also contribute to compensate for ventricular heterogeneity. In fact, it is important to note that eel ventricular myocardiocytes of both the spongiosa and the compacta express an ‘eNOS-like’ isoform [for terminology and references in fish, see Imbrogno et al. (Imbrogno et al., 2011)] that, by linking multiple extracellular signals that converge to regulate eNOS activity in the cells, acts as a spatio-temporal integrator of the heart function, as discussed below.
The bulbus arteriosus of the eel shows, in the inner wall, an irregular surface because of the presence of branching ridges, covered by flattened EE cells that contain dense bodies with a secretory function (Icardo et al., 2000). The middle layer is formed by smooth muscle cells, embedded in a meshwork matrix containing thin and thick filaments (Icardo et al., 2000). The stretching of this meshwork suggests an active role as smooth muscle in wall dynamics. Furthermore, large areas of the extracellular space are occupied by elastin-like materials, whose amount decreases toward the external layer (Icardo et al., 2000). On the contrary, the collagen, although present across the entire wall thickness, increases from the inner toward the outer bulbus surface. Conceivably, such a gradient of collagen matrix may increase wall strength, maintaining bulbus dilation within safe physiological changes.
The myocardium is covered by an external mesothelial layer, the epicardium. It is formed by flattened cells containing abundant pinocytotic vesicles, which is consistent with an active solute interchange with the pericardial cavity (Icardo et al., 2000). As in other teleosts, the detection of epicardial natriuretic peptide (NP) receptors (Cerra et al., 1996) suggests an NP-dependent control of pericardial activity. Moreover, the presence of an ‘eNOS-like’ enzyme at the epicardial level (Amelio et al., 2006; Amelio et al., 2008; Imbrogno et al., 2011) indicates an autocrine–paracrine function in relation to both interstitial fluid balance and myocardial remodeling/regeneration (for details, see Poss et al., 2002; Lepilina et al., 2006).
Regulation of cardiac performance: from organ to molecular level
Thanks to their adaptive and acclimatory abilities to face changes in environmental salinity, extreme temperatures, oxygen availability, sustained enforced activity, etc. (see below), eels are well suited for investigating the mechanisms involved in the modulation of the heart function.
Regulation of cardiac output (CO), i.e. the product of heart rate (HR) and stroke volume (SV), is obtained through both intrinsic and extrinsic (neuro-humoral) mechanisms. In fish, as well as in other vertebrates, hemodynamic loads (filling pressure or preload,
B A
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and systemic aortic pressure or afterload) are primary determinants of CO. The neuro-humoral control is mainly achieved through the adrenergic and cholinergic innervation, and a variety of humoral agents, including cardiac NPs, angiotensin II (Ang II) and catecholamines (CAs) released by both extracardiac and cardiac chromaffin cells (Randall and Perry, 1992; Farrell and Jones, 1992; Nilsson and Holmgren, 1992).
Intrinsic regulation: the Frank–Starling response
The intrinsic regulation of cardiac function is exemplified by the length-dependent response to changing preload. In mammals, the myocardial response to increased preload involves a rapid increase in the force of contraction (i.e. the Frank–Starling response), attributed to a length-dependent increase in cross-bridge formation and myofilament calcium responsiveness (Katz, 2002), followed by a slower and a less pronounced increase in inotropy (i.e. the Anrep effect), correlated with an increased intracellular Ca2+ transient
(Calaghan et al., 1999; Casadei and Sears, 2003). Although it is not yet clear whether in fish the cardiac response to stretch assumes the same biphasic module, the key role of the preload-induced increases in developed force has been recognized in various species of teleosts, including the eel (Tota et al., 1991; Farrell and Jones, 1992; Dunmall and Schreer, 2003; Icardo et al., 2005; Imbrogno et al., 2011; Amelio et al., 2013), and has been attributed to a great myocardial extensibility of the highly trabeculated hearts (Shiels and White, 2008). However, several studies have proposed that increased HR is the primary means of increasing CO during swimming (see Axelsson et al., 1994; Altimiras and Larsen, 2000; Cooke et al., 2003), suggesting that the relative balance of HR versusSV for adjusting CO in fish tightly depends on species-related differences as well as on the kind of the experimental design employed.
We have reported that the high sensitivity of the A. anguillaheart to preload increases is significantly enhanced by a basal release of
endogenous NO (Imbrogno et al., 2001). This involves a protein kinase B (Akt)-mediated activation of ‘eNOS-like’-dependent NO production, which, acting through a non-classical cGMP-independent pathway, induces a phospholamban (PLN)-S-nitrosylation-dependent increase of sarcoplasmic reticulum (SR)-Ca2+reuptake (Fig.2) (Garofalo et al., 2009; Cerra and Imbrogno,
2012).
Extrinsic regulation: the neurohumoral control Innervation
Aneural in the hagfish or only innervated by cholinergic fibers in lampreys and elasmobranchs, in teleosts the heart has an established sympathetic and parasympathetic innervation through the ‘vagosympathetic’ trunk (Laurent et al., 1983; Nilsson, 1983; Taylor, 1992). The eel heart, like that of other teleosts, receives both adrenergic and cholinergic innervation, whose importance varies according to species-specific characteristics and circumstances. However, because of the lack of longitudinally connected sympathetic chains, the teleost sympathetic nervous system (SNS) is less developed than that of birds and mammals (Laurent et al., 1983; Taylor, 1992). The peripheral nerves are replaced by aggregates of CA-containing chromaffin cells, which in many species become components of the diffuse neuroendocrine tissue (Burnstock, 1969). These chromaffin cells, often associated with sympathetic nerves and/or cholinergic inputs, provide a zonal CA production, thereby contributing to the humoral cardiovascular regulation (Gannon and Brunstock, 1969; Abrahamsson et al., 1979; Nilsson and Holmgren, 1992; Tota et al., 2010).
Acetylcholine
Acetylcholine (ACh), released by cholinergic fibers carried in the vagus, are responsible for lowering HR, and several studies in teleosts showed an abolition of bradycardia by vagotomy or Caveolin-I eNOS
Stretch
NO
PLN SERCA SERCA-Ca2+
Heterometric autoregulation (Starling response)
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0 20 40 60 80 100 120 140 160 180
200 Control Starling
Optical density
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30 kDa12 kDa
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Biotin switch assay
PLN PLN nitrosylation
atropine injection (e.g. Saito, 1973). Differences in the ACh-dependent control of HR in teleosts have been reported in relation to temperature. For example, the level of cholinergic inhibition of HR is greater in cold-acclimated rainbow trout with respect to warm-acclimated rainbow trout (Wood et al., 1979). In contrast, the level of cholinergic inhibition of HR is lower in cold-acclimated A. anguilla(Seibert, 1979). Moreover, in several teleost species, an ACh-dependent reduction of contractility has been reported (Randall, 1970; Holmgren, 1977; Cameron and Brown, 1981).
Cardiac cholinergic stimuli are mediated by muscarinic receptors (mAChRs) whose relative amounts vary among species and tissues. Five different mAChRs (M1-M5; Brodde and Michel, 1999) have been described in vertebrates. M2 and M4 subtypes, preferentially located on the myocardiocytes and principally coupled to adenylate cyclase inhibition, reduce intracellular cAMP levels and decrease the L-type Ca2+current, thereby eliciting negative chronotropic and
inotropic effects [for references, see Hove-Madsen et al. (Hove-Madsen et al., 1996) and Gattuso et al. (Gattuso et al., 1999)]. M1, M3 and M5 subtypes, largely located on the vascular endothelial and EE cells, and functionally coupled to intracellular Ca2+
mobilization via phospholipase C, phospholipase A2 and phospholipase D, mediate positive cholinergic inotropism (Brodde and Michel, 1999). In A. japonica(Chan and Chow, 1976) and A. anguilla (Imbrogno et al., 2001), exogenous ACh induces a biphasic inotropic effect. In A. anguilla, the positive inotropism is mediated by M1 receptors while the negative inotropism involves M2 subtypes (Imbrogno et al., 2001); the M1-dependent positive inotropism occurs through an NO-cGMP signal transduction mechanism and is abolished when the EE is functionally damaged (Imbrogno et al., 2001). The EE-dependent transduction of M1-mediated cholinergic response suggests that in the eel this receptor subtype is located at the EE level (Fig.3), resembling the situation in mammals. Of note, the positive cholinergic response has been mostly observed in the spring (Imbrogno et al., 2001), supporting the idea that seasonal factors can fine-tune the sensitivity of the eel heart to cholinergic regulation.
Catecholamines
CAs reach cardiac adrenoceptors (ARs) via the circulation (adrenaline and noradrenaline) and the SNS (noradrenaline) terminals. In teleosts, basal plasma CAs levels are low, whereas the nervous activity plays a major role (Smith, 1978; Holmgren and
Nilsson, 1982; Smith et al., 1985; Axelsson, 1988; Randall and Perry, 1992). While in most teleost species, circulating CAs increase in response to various physical and environmental stimuli (exhaustive exercise, hypoxia, hypercapnia, etc.), the eel does not exhibit increased plasma CAs or cortisol, an indicator of sublethal chronic stress, in response to stressful stimuli (Wendelaar Bonga, 1997). Thus, the members of the genus Anguillaare considered stress-tolerant animals (McKenzie et al., 2003).
The heart response to CA stimulation is mediated by α- and β -ARs and their subtypes, identified in fish, as well as in other vertebrates (Ask, 1983). The effects of adrenergic stimulation have been studied in various teleost species [e.g. A. anguilla(Forster, 1976; Peyraud-Waitzenegger et al., 1980; Pennec and Peyraud, 1983; Pennec and Le Bras, 1984; Pennec and Le Bras, 1988) and Anguilla dieffenbachti (Forster, 1981)]. For example, a basal adrenergic excitatory tone, which prevails over the cholinergic tone, has been described by Pennec and Le Bras (Pennec and Le Bras, 1984). However, more recent studies reported considerably higher cholinergic than adrenergic tones in various species of fish [for references, see Sandblom and Axelsson (Sandblom and Axelsson, 2011)]. Peyraud-Waitzenegger et al. (Peyraud-Waitzenegger et al., 1980) have correlated the cardiac adrenoceptors function in teleosts to seasonal changes in temperature. Moreover, an adrenergic-dependent increase of both HR (Graham and Farrell, 1989) and the Frank–Starling response (Farrell et al., 1986) has been proposed; however, the specific mechanisms underpinning these, as well as other, CA-elicited cardiac actions remain to be clarified.
A re-evaluation of these studies in light of the present knowledge arouses some theoretical and methodological considerations (Randall and Perry, 1992). In fact, the cardiac response to AR stimulation depends not only on the species-related differences and the organizational level under study (i.e. in vivo cardiovascular system versusisolated and denervated working heart), but also on the stress impact and the consequent compensatory responses (e.g. divergent stress coping styles). In the context of this rapidly growing area of fish physiology (Winberg and Nilsson, 1993; Cossins et al., 2006; Johnson et al., 2006), it has become crucial to appreciate that the CAs under study are, at the same time, strikingly implicated in the stressed condition of the laboratory animal. As discussed by Epple and Brinn (Epple and Brinn, 1987), another limitation has been the use of pharmacological but not
physiological concentrations of CAs. This may have delayed the identification of the now-acknowledged fine-tuned yin–yang modulation exerted by CAs as components of the complex AR–G-protein-coupled signal transduction pathways, including NO-cGMP (see below).
In more recent years, the identification of a new type of cardiac
β-AR, β3, gave new insights into the adrenergic control of the teleost heart function. As well as β1- and β2-ARs, β3-AR belongs to the G-protein coupled receptors characterized by seven transmembrane domains of 22–28 amino acids. In mammals, β3 activation elicits negative inotropism and lusitropism (Gauthier et al., 2000; Angelone et al., 2008a), involving NO-cGMP signaling. Similarly, in the eel, β3-AR activation decreases cardiac mechanical performance through a pertussis-toxin-sensitive Gi protein mechanism, consistent with a major β3-AR myocardial localization, and requires the NO-cGMP-cGMP-activated protein kinase G (PKG) cascade (Fig.3) (Imbrogno et al., 2006). Furthermore, isoproterenol (ISO) stimulation, in addition to its classic positive inotropism, induces a negative inotropism possibly mediated by β3-AR (Imbrogno et al., 2006). The discovery of β 3-ARs and their involvement in counteracting ISO stimulation in the eel heart has not only contributed to clarify the adrenergic versatility that controls its function, but has also shed new light on the role of the balanced cardiac regulation of β1/β2 and β3-AR systems in fish.
Angiotensin II
Angiotensin II (Ang II), the principal effector of the renin-angiotensin system, is a pluripotential hormone whose wide biological actions have been extensively studied both in mammalian and non-mammalian vertebrates, including fish (see Kobayashi and Takei, 1996).
Ang II mediates its cardiac effects via binding to plasma membrane AT1 and AT2 receptors, classified according to their
selectivity for specific ligands [see references in de Gasparo and Cerra et al. (de Gasparo, 2002; Cerra et al., 2001)]. AT1 is
responsible for most of the Ang II-mediated effects on cardiac performance (i.e. chronotropism and inotropism) and rate of protein synthesis in isolated myocyte preparations [for references, see Imbrogno et al. (Imbrogno et al., 2003)]. In contrast, cardiac AT2
receptor antagonizes AT1growth promoting effects viaactivation
of a number of phosphatases. This receptor is also coupled with the NO-cGMP signaling, either directly, or indirectly, through enhanced bradykinin or eNOS expression (Dostal, 2000). Several fish Ang II receptors have been cloned (Marsigliante et al., 1996; Tran van Chuoi et al., 1998). For example, in the eel, an angiotensin receptor cDNA sequence [GenBank accession number AJ05132 (Tran van Chuoi et al., 1998)] shows 60% homology with the mammalian AT1receptor (Russell et al., 2001).
Most of the Ang II-mediated cardiovascular effects documented in teleosts appear to be species specific. For example, while Ang II injected in trout produces a hypertension-dependent reflex bradycardia with reduced CO [for references, see Russell et al. (Russell et al., 2001)], in the American eel, Anguilla rostrata, the Ang II-induced hypertension depends on the increased CO rather than on changes in systemic resistances (Butler and Oudit, 1995; Oudit and Butler, 1995). In the eel A. rostrata, as well as in the trout Oncorhynchus mykiss, Ang II exerts both direct and indirect (i.e. viacardiac adrenoceptors) cardiac stimulatory effects (Oudit and Butler, 1995; Bernier et al., 1999). Conversely, in the European eel, A. anguilla, Ang II exerts a direct cardioinhibition involving AT1-like receptors, Gi/o proteins and an EE-NO-cGMP cascade
(Imbrogno et al., 2003) (Fig.3). It has been proposed that the cardio-suppressive effect observed in the in vitroeel (A. anguilla) heart could function as a local cardio-inhibitory protection against systemic cascades of convergent excitatory stimuli targeting the heart (Imbrogno et al., 2003).
Chromogranin A-derived peptides
Chromogranin A (CgA) is the major member of the chromogranin/secretogranin family of glycoproteins expressed in all neuroendocrine cells (Winkler and Fischer-Colbrie, 1992). As first shown in the adrenal medulla, it is co-stored and co-released with CAs from the secretory vesicles of the chromaffin cells (Helle et al., 2007). CgA is the precursor of several regulatory peptides (i.e. vasostatins, pancreastatin, catestatin and parastatin), generated by cell-, tissue- and species-specific proteolytic processes, most of which act as powerful inhibitors of endocrine secretion [see Helle et al. (Helle et al., 2007) and references therein].
Evidence from the past decade suggests that CgA-derived vasostatin 1 (VS-1) and catestatin (Cts) peptides represent fundamental players in the cardiovascular homeostasis, contributing to the humoral regulation of the vertebrate hearts (Mazza et al., 2010). The teleost heart is not an exception. In fact, recent evidence in A. anguillashowed that both VS-1 and Cts exert cardio-suppressive actions and counteract adrenergic-mediated positive inotropism (Imbrogno et al., 2004; Imbrogno et al., 2010). In particular, the VS-mediated negative inotropism is abolished by the pre-treatment with Ca2+ and K+ channel antagonists, and
involves a NO-mediated pathway, as well as muscarinic and adrenergic receptors (Imbrogno et al., 2004; Tota et al., 2004). Moreover, cytoskeleton integrity is a prerequisite for the VS-induced negative inotropism, the latter being blocked by inhibitors of the cytoskeletal dynamics (Mazza et al., 2007). Similarly, the Cts-dependent cardiosuppressive action observed in the eel involves the adrenergic system and occurs via an NO-cGMP cascade (Imbrogno et al., 2010). In addition, Cts significantly increases the Frank–Starling response of the eel heart through a mechanism that involves NO and SR-CA2+ATPase (SERCA2a)
pumps (Imbrogno et al., 2010).
A considerable number of studies performed in teleosts show that under stress challenges, such as hypoxia, anemia, acidosis, hypercapnia, exhaustive exercise and physical disturbances, there is a sudden release of CAs from chromaffin cells, such as those richly embedded into the walls of the posterior cardinal vein close to the head kidney (Nandi, 1961). Accordingly, the heart may become targeted at the same time by these blood-borne CAs and those released by both SNS terminals and cardiac chromaffin cells (Nilsson and Holmgren, 1992; Farrell and Jones, 1992), which might become harmful in the absence of local counter-regulatory mechanisms. As comparatively proposed in frog (Mazza et al., 2008) and rat (Angelone et al., 2008b) hearts, CgA may function as prohormone for inhibitory proteins that, if released under stressful conditions, might protect the organ from excessive excitatory stimuli, acting as anti-adreno-sympathetic stabilizers (Helle et al., 2007). It remains to be established whether and to what extent CgA and its derived peptides contribute to the stress resistance of the eel.
Natriuretic peptides
and ventricular natriuretic peptide (VNP)] (Takei et al., 1991; Takei and Balment, 1993; Evans, 1995) were subsequently found in the hearts of a large number of non-mammalian vertebrates, including fish (Netchitailo et al., 1987; Takei et al., 1990; Bjenning et al., 1992; Larsen et al., 1994; Kawakoshi et al., 2003). With the exception of a few species – medaka (Oryzias latipes), which does not possess ANP, and eel (Anguilla japonica), which lacks BNP (Takei et al., 1991; Takei et al., 1994a; Takei et al., 1994b) – all NPs are expressed in teleosts. Recently, BNP was also detected in the rainbow trout (Oncorhynchus mykiss) (Johnson and Olson, 2009).
Four types of NP receptors (NPRs), named NPRA, NPRB, NPRC and NPRD, are present from cyclostomes to mammals [for references, see Takei et al. (Takei et al., 2011)]. In particular, NPRC-like receptors are expressed in the atrial and ventricular myocardium of the eel (A. anguilla), while the ventricular EE appears to express an NPRA-type receptor that is able to bind ANP and VNP with almost equal affinity (Cerra et al., 1996). A receptor with high CNP affinity, presumably NPRB, was also reported in the endothelial and epicardial layers of the bulbus arteriosus of A. anguilla (Cerra et al., 1996), where it may mediate a CNP-dependent modulation of the bulbar hemodynamics (e.g. the Windkessel function) (see Icardo et al., 2000).
In all vertebrates, the primary stimulus for acute atrial NP release is the hypervolemia-induced stretch (Ruskoaho, 1992). Once released, NPs activate complex signaling networks that control heart–vessel and volume–ion homeostasis at various levels [for references, see Tota et al. (Tota et al., 2010)]. These networks directly coordinate chronotropic, inotropic and vasorelaxant responses, and indirectly minimize cardiac hemodynamic loads. The hypothesis that NPs also exert a cardioprotective action from excessive preloads and afterloads in fish is supported by evidence obtained in various species (see Tota et al., 2010). The finding that the transfer from freshwater to seawater of the Japanese eel, A. japonica, transiently increases both ANP and VNP (Kaiya and Takei, 1996a) suggests that an osmotic mechanism is also able to regulate the NP release from the teleost hearts. Although volemic stimuli are also stimulatory for NP release in eel and trout, hyperosmolality may be more potent than hypervolemia in stimulating atrial ANP release (Kaiya and Takei, 1996b; Cousins and Farrell, 1996). Moreover, in marine eels, ANP is antidipsogenic at doses as low as non-hypotensive (Tsuchida and Takei, 1998). These observations indicate a situation in fish opposite to that found in mammals, in which blood volume decrease inhibits ANP secretion and the ANP-dependent hemodynamic actions appear dominant if compared with the osmoregulatory effects.
Autocrine–paracrine regulation: the role of the NOS/NO system
Recent evidence in fish has highlighted the role played by cardiac autocrine–paracrine factors in modulating the whole-ventricle function, widening the concept of heart autoregulation and neurovisceral integration also to these vertebrates (for a review, see Tota et al., 2010). In particular, the role of NO as a major autocrine/paracrine organizer of complex connection-integration signals has been recently assessed in the fish heart (Imbrogno et al., 2010). NOS isoenzymes [i.e. endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS)], located in almost every heart tissue, regulate, through their distinct spatial subcellular compartmentation, the production of NO close to its molecular targets (Seddon et al., 2007). NO, generated in one cell, can act on the adjacent cells (paracrine modulation) or on the cell
itself (autocrine modulation) (Moncada et al., 1991). A hallmark of the paracrine NO signal, best evaluated in the avascular, or poorly vascularized, fish hearts (Gattuso et al., 2002; Imbrogno et al., 2011), is represented by the EE-induced contractile modulation of myocardial performance. Under basal conditions, the EE-generated NO exerts cGMP-dependent negative contractile influences. In contrast, the functional damage of the EE, with the consequent interruption of the EE-NOS-cGMP axis, elicits positive contractile effects (Imbrogno et al., 2010). This EE-eNOS signaling, located at the crossroads of many extrinsic and intrinsic neuro-endocrine pathways, coordinates many chemically activated cascades. For example, endoluminal chemical stimuli such as ACh, Ang II, VS-1, Cts, as well as β3 adrenoceptor activation, all converge in their contractile effects viaNO signalling, which requires the obligatory involvement of the EE (Imbrogno et al., 2001; Imbrogno et al., 2003; Imbrogno et al., 2004; Imbrogno et al., 2006; Imbrogno et al., 2010). Therefore, the EE not only represents a quantitatively important source of NO, but it is also necessary for coupling the intracavitary stimuli to the eNOS/NO system.
More recently, an NO-dependent autocrine pathway, related to the specific subcellular localization and regulation of the different NOS isoforms, as well as to NO target proteins, has also been described [for references, see Seddon et al. (Seddon et al., 2007)]. Importantly, such subtle spatial compartmentation and signaling network coordination between cardiac NO and specific intracellular effectors (Hare, 2003) allow NO to achieve local control of different cellular functions (Iwakiri et al., 2006). At the same time, a relatively high local concentration of NO equivalents may be generated, which in turn provides a favorable environment for protein S-nitrosylation (Lima et al., 2010). Of note, an NO-induced modulation of the heterometric regulation has been shown in the eel. In fact, Garofalo and co-workers (Garofalo et al., 2009) have recently reported that the ‘beat-to-beat’ regulation of the in vitro working heart of the eel A. anguilla is directly modulated by myocardial autocrine NO that, through a regulation of PLN S-nitrosylation-dependent calcium reuptake by SERCA2a pumps, modulates myocardial relaxation. This evidence supports the notion that the NO ability to modulate ventricular performance through NOS isoform compartmentation, as well as differences in their mode of stimulation and recruitment of distinct downstream pathways, was indeed a crucial and early event during the vertebrate evolution.
Nitrite as a bioactive NO source
Nitrite (NO2−) represents a key intrinsic signaling molecule in many
additional direct uptake of exogenous nitrite from the environmental water across the respiratory surfaces (Jensen, 2009). Therefore, fish need to balance the advantageous access to an ambient pool of nitrite for internal NO production with the potentially dangerous effects of nitrite-polluted habitats (see Jensen and Hansen, 2011).
In the eel, nitrite represents a significant source of bioactive NO with consequent influences on heart function. It negatively affects the basal cardiac mechanical performance through an NOS-dependent mechanism, which involves a cGMP/PKG transduction signaling (Cerra et al., 2009). Moreover, nitrite profoundly influences the Frank–Starling response through an NO/cGMP/PKG pathway and S-nitrosylation of both membrane and cytosolic proteins (Angelone et al., 2012).
Eel growth and cardiac morphodynamics
A phenomenon characteristic of fish is indeterminate growth, as a consequence of which, unlike mammals, the organism can grow throughout most of adulthood. In fish, the cardio-circulatory system is exposed to dramatic phylogenetic and ontogenetic rearrangements, as well as to severe environmental stresses, that are matched by plastic changes of the major heart pump, the ventricle. These can be attained through cardiomyocyte hypertrophy and/or hyperplasia of both the compacta and the spongiosa (Gamperl and Farrell, 2004).
Among teleosts, the eel A. anguillaprovides a striking example of growth-related plasticity of ventricular structure and function. By comparing small juvenile fish with their large adult counterparts, Cerra et al. (Cerra et al., 2004) evaluated from organ to cellular and sub-cellular levels the ventricular morphodynamic changes occurring during growth. The two groups show similar responses at increasing filling pressure (preload), but remarkably differ at increasing afterload (Fig.4A). The latter reflects the cardiac ability to adjust pressure development in response to increased peripheral resistances, as in the case of intense exercise. Small eel hearts decreased CO at afterloads greater than 3kPa, in contrast to largest hearts, which maintained constant CO up to 6kPa (Fig.4A). These changes in mechanical performance are related to structural differences. In fact, if compared with small eels, large eels show an increase in the compacta thickness and in the diameter of the trabeculae in the spongiosa, together with a reduction of the lacunary spaces (Fig.4Bi,iv). The increased compacta thickness is attained through an enlargement of both muscular and vascular compartments and a reduction of the interstitium; consequently, this layer appears more compacted (Fig.4Bii,v). Therefore, because of these growth-related morphodynamic changes, the cardiac ventricle of small eels, with its limited response to pressure overload and large lacunary spaces, appears better suited to produce volume work, while that of the large eels is better adapted to produce pressure work [for references, see Icardo et al. (Icardo et
i ii iii
iv v vi
A
40B
0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
Preload (kPa) Afterload (kPa)
* * *
*
*
*
*
*
*
*
*
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*
*
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*
*
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*
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CO (ml min
–1
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–1
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20
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al., 2005)]. Myocardial hyperplasia in both the compacta and the spongiosa is indicated by the higher number of myocytes associated with a reduced cross-sectional area and myofibrillar compartment. However, morphometric and ultrastructural analysis revealed that the growth pattern of the two layers is slightly different. Compacta and spongiosa myocytes become smaller during growth, reducing their myofibrillar compartment, a reduction that is threefold smaller in the compacta than in the spongiosa. Moreover, unlike the spongiosa, the compacta shows an increase in the mitochondrial compartment (Fig.4Biv,vi), which appears bio-energetically important for the myofibrillar apparatus to match the increased basal HR and the higher pumping capacity of large eels (Cerra et al., 2004). The possible redox-dependent signalling mechanisms and transcription factors known to orchestrate the increase in mitochondrial content, which is associated with workload-induced cardiac growth, have been discussed in mammals (Leary et al., 2002) and in fish (Birkedal et al., 2006; Urschel and O’Brien, 2008).
Cardiac responses to environmental challenges
Temperature
Fish possess a remarkable cardio-circulatory adaptability to seasonal thermal changes (Hazel and Prosser, 1974; Cossins and Bowler, 1987) and, like other ectotherms, they can compensate for the direct effects of temperature on physical processes and enzymatic reaction kinetics through temperature acclimation or acclimatization (for reviews, see Driedzic and Gesser, 1994; Gamperl and Farrell, 2004). Moreover, in addition to regular and predictable temperature fluctuations, such as those related to season, many fish species also experience acute thermal changes (e.g. fish vertical movements in the water column across thermoclines, or diurnal temperature changes in shallow streams) [salmon (Brett, 1971); blue marlin (Block et al., 1992); tuna (Block et al., 1997); trout (Matthews and Berg, 1997; Reid et al., 1997)] that may cause significant and rapid changes of body temperature with immediate effects on the heart function. For example, in many non-polar species, cold acclimation is associated with an increased heart size, which, in turn, increases resting SV and lowers HR (Driedzic et al., 1996). In various species of teleosts, including rainbow trout and carp, acute changes in temperature affect cardiac contractility, as well as HR, which is inversely related with the isometric force produced (Shiels et al., 2002; and references therein). In migrating eels, which face acute temperature fluctuations while performing vertical migration, diving into colder waters (~6–8°C) during the day and ascending to shallow warmer waters (12–14°C) at night (Aarestrup et al., 2009), the consequent increases of ventricular power production can compensate for the cooling-induced upper limit placed on HR (Methling et al., 2012). The relationship between cholinergic tone and temperature has been mentioned above.
Interestingly, a temperature-sensitive paradigm of subtle heart regulation has been recently shown in A. anguillaby Amelio and co-workers (Amelio et al., 2013). In fact, the previously mentioned NO-mediated modulation of the Frank–Starling response appears temperature dependent, as it is preserved during the physiological acclimation at different temperatures, but is abolished by acute thermal shock conditions. These results are paralleled by a thermal-related decrease of both activated ‘eNOS-like’ and pAkt expression, the latter being a major signaling protein kinase phosphorylating eNOS in vitroas well as in vivo(Dimmeler et al., 1999). This newly discovered thermal sensitivity of the
NOS/NO-dependent modulation of the Frank–Starling response provides another example of the fascinating plasticity of the eel heart.
Exercise
An extensive body of literature focuses on the metabolic, respiratory and cardiovascular adjustments during swimming (Randall, 1982; Randall and Perry, 1992; McKenzie et al., 2004). Swimming in fish is generally classified as either burst or sustained (Tierney, 2011). While burst swimming is essentially an anaerobic activity of white muscle, often decreasing CO, sustained swimming is aerobic, increasing both CO, through changes in HR and SV, and ventilation (Stevens and Randall, 1967; Kiceniuk and Jones, 1977). However, eels may represent an exception. In fact, unlike most teleosts, in the Australian eel A. australis, sustained swimming does not increase CO but, because of branchial vessel constriction, instead increases ventral aortic blood pressure without changing dorsal aortic pressure (Davie and Foster, 1980). The significance of these changes remains to be clarified.
Hypoxia
Fish employ various physiological strategies to regulate the rates of O2 uptake and aerobic metabolism relatively independent of
water O2availability. For example, in most teleosts, acute hypoxia
produces a reflex cholinergic bradycardia, which contributes to the regulation of O2uptake [for references, see Iversen et al. (Iversen
et al., 2010)]. Eels possess a great ability to regulate the aerobic metabolism in hypoxia. This has been correlated to the exceptional capacity of their cardiovascular and ventilatory systems to meet the O2demands of their tissues (McKenzie et al., 2002). Under hypoxic
conditions, eels do not display a significant bradycardia until PO2 declines at levels well below the critical PO2 (i.e. the PO2values below which aerobic metabolic rate is directly limited by, and dependent upon, O2levels in the water). This suggests that reflex
bradycardia does not contribute to maintenance of oxygen consumption and regulation of standard metabolic rate (Iversen et al., 2010). It has been reported that, unlike other teleost species (e.g. salmonids), in the exercising eels, the capacity for O2convection
is not closely matched to maximum tissue O2 demands, but can
exceed them [for references, see McKenzie et al. (McKenzie et al., 2003)]. Thus, it is conceivable that the aerobic scope and active metabolic rate are determined by the maximum capacity of the tissues to use O2 and perform work. However, under anaerobic
conditions, the eel heart needs an extracellular glucose supply to maintain force/pressure development. In fact, it has been demonstrated that under anoxia, isolated perfused eel hearts maintain pressure development over 2h of perfusion if glucose is supplied in the medium; on the contrary, in the absence of glucose, hearts perfused with the anoxic medium fail within 30min (Bailey et al., 2000).
Hypercapnia
Another example of the remarkable capacity for physiological adaptation that characterizes the genus Anguillais its exceptional tolerance to very severe chronic hypercapnia (Larsen and Jensen, 1997). Severe levels of hypercapnia, with a consequent reduction of intracellular pH, interfere with cellular metabolism through effects on the function of pH-sensitive proteins (Heisler, 1984; Heisler, 1993). In the eel, unlike active teleosts such as salmonids, elevated water CO2levels do not influence exercise performance
2002). How and to what extent the eel myocardium is able to compensate its intracellular pH to preserve cardiac performance represents a challenge for future studies.
Conclusions
This review emphasizes the amazing flexibility of the eel heart in relation not only to the basic cardiac design and elaborated neuro-endocrine traits, but also to its particular biochemical-metabolic plasticity and acclimatory potentialities. The eel heart appears to be fine-tuned by intrinsic (hemodynamic forces), extrinsic (humoral agents) and environmental (temperature, exercise, etc.) factors that, downstream from their stimulation targets, activate complex molecular signal-transduction networks. More recent evidence has shown the major role played by the NOS/NO system as a paracrine–autocrine modulator able to interact with a number of intracellular targets (autocrine action), and with sites of signal integration of other transduction pathways (paracrine action).
In a comparative perspective, the information reported here provides useful tools with which to not only identify cardiac structural designs and complex modulatory neuro-humoral networks, but also understand the putative relationship between the evolutionary selective pressures provided by the life history of these animals and their peculiar physiological features (e.g. the exceptional tolerance to chronic hypercapnia, the notable capacity to meet the O2demands of routine and active metabolism, and the
remarkable cardio-circulatory adaptability to thermal changes). In addition, this information may contribute to deciphering how the heterogeneities of cardiac form and function support the uniformity of the whole heart pumping activity. Further studies will clarify to what extent the physiological characteristics revealed in the eel heart may highlight general traits of cardiac design that are of universal significance or specific attributes of this fascinating group of teleosts.
List of abbreviations
ACh acetylcholine
Ang II angiotensin II
ANP atrial natriuretic peptide
AR adrenoceptor
BNP brain natriuretic peptide
CA catecholamine
CgA chromogranin A
CNP C-type natriuretic peptide
CO cardiac output
Cts catestatin
EE endocardial endothelium
eNOS endothelial NOS
HR heart rate
iNOS inducible NOS
ISO isoproterenol
mAChR muscarinic receptor
nNOS neuronal NOS
NO nitric oxide
NOS nitric oxide synthase
NP natriuretic peptide
NPR natriuretic peptide receptor
PKG protein kinase G
PLN phospholamban
SNS sympathetic nervous system
SV stroke volume
VNP ventricular natriuretic peptide
VS-1 vasostatin 1
Acknowledgements
The author thanks Dr Daniela Amelio for support with morphological characterization of eel hearts.
Competing interests No competing interests declared.
Funding
This study was supported by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca).
References
Aarestrup, K., Okland, F., Hansen, M. M., Righton, D., Gargan, P., Castonguay, M., Bernatchez, L., Howey, P., Sparholt, H., Pedersen, M. I. et al.(2009). Oceanic spawning migration of the European eel (Anguilla anguilla). Science325, 1660.
Abrahamsson, T., Holmgren, S., Nilsson, S. and Pettersson, K.(1979). On the chromaffin system of the African lungfish, Protopterus aethiopicus. Acta Physiol. Scand.107, 135-139.
Altimiras, J. and Larsen, E.(2000). Non-invasive recording of heart rate and ventilation rate in rainbow trout during rest and swimming. Fish go wireless! J. Fish Biol.57, 197-209.
Amelio, D., Garofalo, F., Pellegrino, D., Giordano, F., Tota, B. and Cerra, M. C.
(2006). Cardiac expression and distribution of nitric oxide synthases in the ventricle of the cold-adapted Antarctic teleosts, the hemoglobinless Chionodraco hamatusand the red-blooded Trematomus bernacchii. Nitric Oxide15, 190-198.
Amelio, D., Garofalo, F., Brunelli, E., Loong, A. M., Wong, W. P., Ip, Y. K., Tota, B. and Cerra, M. C.(2008). Differential NOS expression in freshwater and aestivating Protopterus dolloi (lungfish): heart vskidney readjustments. Nitric Oxide18, 1-10.
Amelio, D., Garofalo, F., Capria, C., Tota, B. and Imbrogno, S.(2013). Effects of temperature on the nitric oxide-dependent modulation of the Frank-Starling mechanism: the fish heart as a case study. Comp. Biochem. Physiol.164A, 356-362.
Angelone, T., Filice, E., Quintieri, A. M., Imbrogno, S., Recchia, A., Pulerà, E., Mannarino, C., Pellegrino, D. and Cerra, M. C.(2008a). Beta3-adrenoceptors modulate left ventricular relaxation in the rat heart viathe NO-cGMP-PKG pathway. Acta Physiol. 193, 229-239.
Angelone, T., Quintieri, A. M., Brar, B. K., Limchaiyawat, P. T., Tota, B., Mahata, S. K. and Cerra, M. C.(2008b). The antihypertensive chromogranin a peptide catestatin acts as a novel endocrine/paracrine modulator of cardiac inotropism and lusitropism. Endocrinology149, 4780-4793.
Angelone, T., Gattuso, A., Imbrogno, S., Mazza, R. and Tota, B.(2012). Nitrite is a positive modulator of the Frank-Starling response in the vertebrate heart. Am. J. Physiol.302, R1271-R1281.
Aoyama, J., Hissmann, K., Yoshinaga, T., Sasai, S., Uto, T. and Ueda, H.(1999). Swimming depth of migrating silver eels Anguilla japonica released at seamounts of the West Mariana Ridge, their estimated spawning sites. Mar. Ecol. Prog. Ser.186, 265-269.
Ask, J. M.(1983). Comparative aspects of adrenergic receptors in the heart of lower vertebrates. Comp. Biochem. Physiol.76A, 543-552.
Axelsson, M.(1988). The importance of nervous and humoral mechanisms in the control of cardiac performance in the Atlantic cod Gadus morhuaat rest and during non-exhaustive exercise. J. Exp. Biol.137, 287-301.
Axelsson, M., Davison, B., Forster, M. and Nilsson, S.(1994). Blood pressure control in the Antarctic fish Pagothenia borchgrevinki. J. Exp. Biol.190, 265-279.
Bailey, J. R., MacDougall, R., Clowe, S. and Driedzic, W. R.(2000). Anoxic performance of the American eel (Anguilla rostrataL.) heart requires extracellular glucose. J. Exp. Zool.286, 699-706.
Bernier, N. J., Mckendry, J. E. and Perry, S. F.(1999). Blood pressure regulation during hypotension in two teleost species: differential involvement of the renin-angiotensin and adrenergic systems. J. Exp. Biol.202, 1677-1690.
Birkedal, R., Shiels, H. A. and Vendelin, M.(2006). Three-dimensional mitochondrial arrangement in ventricular myocytes: from chaos to order. Am. J. Physiol.291, C1148-C1158.
Bjenning, C. E., Takei, Y., Watanabe, T. X., Nakajima, K., Sakakibara, S. and Hazon, N.(1992). A C type natriuretic peptide is a vasodilator in vivo and in vitro in the common dogfish. J. Endocrinol.133, R1-R4.
Block, B. A., Booth, D. and Carey, F. G.(1992). Direct measurements of swimming speed and depth of blue marlin. J. Exp. Biol.166, 267-284.
Block, B. A., Keen, J. E., Castillo, B., Dewar, H., Freund, E. V., Marcinek, D. J., Brill, R. W. and Farwell, C.(1997). Environmental preferences of yellowfin tuna at the northern extent of their range. Mar. Biol.130, 119-132.
Bone, Q., Marshall, N. B. and Blaxter, J. H. S.(1995). Biology of Fishes. Glasgow: Blackie.
Brett, J. R.(1971). Energetic responses of salmon to temperature. A study of some thermal relations in the physiology and freshwater ecology of sockeye salmon (Oncorhynchus nerka). Am. Zool.11, 99-113.
Brodde, O. E. and Michel, M. C.(1999). Adrenergic and muscarinic receptors in the human heart. Pharmacol. Rev.51, 651-690.
Bryan, N. S., Fernandez, B. O., Bauer, S. M., Garcia-Saura, M. F., Milsom, A. B., Rassaf, T., Maloney, R. E., Bharti, A., Rodriguez, J. and Feelisch, M.(2005). Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat. Chem. Biol.1, 290-297.
Burnstock, G.(1969). Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates. Pharmacol. Rev.21, 247-324.
Butler, D. G. and Oudit, G. Y.(1995). Angiotensin-I- and -III-mediated cardiovascular responses in the freshwater North American eel, Anguilla rostrata: effect of Phe8 deletion. Gen. Comp. Endocrinol.97, 259-269.
Cameron, J. S. and Brown, S. E.(1981). Adrenergic and cholinergic responses of the isolated heart of the goldfish, Carassius auratus. Comp. Biochem. Physiol.70C, 109-115.
Casadei, B. and Sears, C. E.(2003). Nitric-oxide-mediated regulation of cardiac contractility and stretch responses. Prog. Biophys. Mol. Biol.82, 67-80.
Cerra, M. C. and Imbrogno, S.(2012). Phospholamban and cardiac function: a comparative perspective in vertebrates. Acta Physiol. 205, 9-25.
Cerra, M. C., Canonaco, M., Takei, Y. and Tota, B.(1996). Characterization of natriuretic peptide binding sites in the heart of the eel Anguilla anguilla. J. Exp. Zool.
275, 27-35.
Cerra, M. C., Tierney, M. L., Takei, Y., Hazon, N. and Tota, B.(2001). Angiotensin II binding sites in the heart of Scyliorhinus canicula: an autoradiographic study. Gen. Comp. Endocrinol.121, 126-134.
Cerra, M. C., Imbrogno, S., Amelio, D., Garofalo, F., Colvee, E., Tota, B. and Icardo, J. M.(2004). Cardiac morphodynamic remodelling in the growing eel (Anguilla anguillaL.). J. Exp. Biol.207, 2867-2875.
Cerra, M. C., Angelone, T., Parisella, M. L., Pellegrino, D. and Tota, B.(2009). Nitrite modulates contractility of teleost (Anguilla anguillaand Chionodraco hamatus, i.e. the Antarctic hemoglobinless icefish) and frog (Rana esculenta) hearts. Biochim. Biophys. Acta1787, 849-855.
Chan, D. K. O. and Chow, P. H.(1976). The effects of acetylcholine, biogenic amines and other vasoactive agents on the cardiovascular functions of the eel, Anguilla japonica. J. Exp. Zool.196, 13-26.
Cooke, S. J., Grant, E. C., Schreer, J. F., Philipp, D. P. and Devries, A. L.(2003). Low temperature cardiac response to exhaustive exercise in fish with different levels of winter quiescence. Comp. Biochem. Physiol.134A, 157-165.
Cosby, K., Partovi, K. S., Crawford, J. H., Patel, R. P., Reiter, C. D., Martyr, S., Yang, B. K., Waclawiw, M. A., Zalos, G., Xu, X. et al.(2003). Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat. Med.9, 1498-1505.
Cossins, A. R. and Bowler, K.(1987). Temperature Biology of Animals. London: Chapman & Hall.
Cossins, A. R., Fraser, J., Hughes, M. and Gracey, A.(2006). Post-genomic approaches to understanding the mechanisms of environmentally induced phenotypic plasticity. J. Exp. Biol.209, 2328-2336.
Cousins, K. L. and Farrell, A. P.(1996). Stretch-induced release of atrial natriuretic factor from the heart of rainbow trout, Oncorhynchus mykiss. Can. J. Zool.74, 380-387.
Crawford, J. H., Isbell, T. S., Huang, Z., Shiva, S., Chacko, B. K., Schechter, A. N., Darley-Usmar, V. M., Kerby, J. D., Lang, J. D., Jr, Kraus, D. et al.(2006). Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood107, 566-574.
Davie, P. S. and Foster, M. E.(1980). Cardiovascular responses to swimming in eels. Comp. Biochem. Physiol.67A, 367-373.
de Bold, A. J., Borenstein, H. B., Veress, A. T. and Sonnenberg, H.(1981). A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci.28, 89-94.
de Gasparo, M.(2002). Angiotensin II and nitric oxide interaction. Heart Fail. Rev.7, 347-358.
Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R. and Zeiher, A. M.(1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature399, 601-605.
Dostal, D. E.(2000). The cardiac renin-angiotensin system: novel signaling mechanisms related to cardiac growth and function. Regul. Pept.91, 1-11.
Driedzic, W. R. and Gesser, H.(1994). Energy metabolism and contractility in ectothermic vertebrate hearts: hypoxia, acidosis, and low temperature. Physiol. Rev.
74, 221-258.
Driedzic, W. R., Bailey, J. R. and Sephton, D. H.(1996). Cardiac adaptations to low temperature in non-polar teleost fish. J. Exp. Biol.275, 186-195.
Dunmall, K. M. and Schreer, J. F.(2003). A comparison of the swimming and cardiac performance of farmed and wild Atlantic salmon, Salmo salar, before and after gamete stripping. Aquaculture220, 869-882.
Duranski, M. R., Greer, J. J., Dejam, A., Jaganmohan, S., Hogg, N., Langston, W., Patel, R. P., Yet, S. F., Wang, X., Kevil, C. G. et al.(2005). Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J. Clin. Invest.
115, 1232-1240.
Ellerby, D. J., Spierts, I. L. Y. and Altringham, J. D.(2001). Slow muscle power output of yellow- and silver-phase European eels (Anguilla anguillaL.): changes in muscle performance prior to migration. J. Exp. Biol.204, 1369-1379.
Epple, A. and Brinn, J. E.(1987). The comparative physiology of the pancreatic islet. In Zoophysiology(ed. D. S. Farner), pp. 61-62. Berlin: Springer-Verlag.
Evans, D. H.(1995). The roles of natriuretic peptide hormones in fish osmoregulation and hemodynamics. Adv. Comp. Environ. Physiol.22, 119-152.
Farrell, A. P. and Jones, D. R.(1992). The heart. In The Cardiovascular System(ed. W. S. Hoar, D. J. Randall and A. P. Farrell), pp. 1-88. San Diego, CA: Academic Press.
Farrell, A. P. and Lutz, P. L.(1975). Apparent anion imbalance in the fresh water adapted eel. J. Comp. Physiol.102, 159-166.
Farrell, A. P., MacLeod, K. R. and Chancey, B.(1986). Intrinsic mechanical properties of the perfused rainbow trout heart and the effects of catecholamines and extracellular calcium under control and acidotic conditions. J. Exp. Biol.125, 319-345.
Forster, M. E.(1976). Effects of catecholamines on the heart and on branchial and peripheral resistance of the eel, Anguilla anguilla(L.). Comp. Biochem. Physiol.55, 27-32.
Forster, M. E.(1981). Effects of catecholamines on the hearts and ventral aortas of the eel, Anguilla australis schmidtiiand Anguilla dieffenbachti. Comp. Biochem. Physiol.70C, 85-90.
Gamperl, A. K. and Farrell, A. P.(2004). Cardiac plasticity in fishes: environmental influences and intraspecific differences. J. Exp. Biol.207, 2539-2550.
Gannon, B. J. and Brunstock, G.(1969). Excitatory adrenergic innervation of the fish heart. Comp. Biochem. Physiol.29, 765-773.
Garofalo, F., Parisella, M. L., Amelio, D., Tota, B. and Imbrogno, S.(2009). Phospholamban S-nitrosylation modulates Starling response in fish heart. Proc. Biol. Sci.276, 4043-4052.
Garofalo, F., Imbrogno, S., Tota, B. and Amelio, D.(2012). Morpho-functional characterization of the goldfish (Carassius auratusL.) heart. Comp. Biochem. Physiol.163A, 215-222.
Gattuso, A., Mazza, R., Pellegrino, D. and Tota, B.(1999). Endocardial endothelium mediates luminal ACh-NO signaling in isolated frog heart. Am. J. Physiol.276, H633-H641.
Gattuso, A., Mazza, R., Imbrogno, S., Sverdrup, A., Tota, B. and Nylund, A.
(2002). Cardiac performance in Salmo salarwith infectious salmon anaemia (ISA): putative role of nitric oxide. Dis. Aquat. Organ.52, 11-20.
Gauthier, C., Langin, D. and Balligand, J. L.(2000). Beta3-adrenoceptors in the cardiovascular system. Trends Pharmacol. Sci.21, 426-431.
Graham, M. S. and Farrell, A. P.(1989). Effect of temperature acclimation and adrenaline on the performance of a perfused trout heart. Physiol. Zool.62, 38-61.
Hare, J. M.(2003). Nitric oxide and excitation-contraction coupling. J. Mol. Cell. Cardiol.35, 719-729.
Hazel, J. R. and Prosser, C. L.(1974). Molecular mechanisms of temperature compensation in poikilotherms. Physiol. Rev.54, 620-677.
Heisler, N.(1984). Acid-base regulation in fishes. In Fish Physiology, Vol. 10A (ed. W. S. Hoar and D. J. Randall), pp. 315-401. New York, NY: Academic Press.
Heisler, N.(1993). Acid-base regulation. In The Physiology of Fishes(ed. D. H. Evans), pp. 343-378. Boca Raton, FL: CRC Press.
Helle, K. B., Corti, A., Metz-Boutigue, M. H. and Tota, B.(2007). The endocrine role for chromogranin A: a prohormone for peptides with regulatory properties. Cell. Mol. Life Sci.64, 2863-2886.
Holmgren, S.(1977). Regulation of the heart of a teleost, Gadus morhua, by autonomic nerves and circulating catecholamines. Acta Physiol. Scand.99, 62-74.
Holmgren, S. and Nilsson, S.(1982). Neuropharmacology of adrenergicneurons in teleostfish. Comp. Biochem. Physiol.72C, 289-302.
Hove-Madsen, L., Méry, P. F., Jurevicius, J., Skeberdis, A. V. and Fischmeister, R.(1996). Regulation of myocardial calcium channels by cyclic AMP metabolism. Basic Res. Cardiol.91Suppl 2, 1-8.
Hyde, D. A., Moon, T. W. and Perry, S. F.(1987). Physiological consequences of prolonged aerial exposure in the American eel, Anguilla rostrata: blood respiratory and acid-base status. J. Comp. Physiol. B157, 635-642.
Icardo, J. M. (2012). The teleost heart: a morphological approach. In Ontogeny and Phylogeny of the Vertebrate Heart(ed. D. Sedmera and T. Wang), pp. 35-53. New York, NY: Springer Science.
Icardo, J. M., Colvee, E., Cerra, M. C. and Tota, B.(2000). Light and electron microscopy of the bulbus arteriosus of the European eel (Anguilla anguilla). Cells Tissues Organs167, 184-198.
Icardo, J. M., Imbrogno, S., Gattuso, A., Colvee, E. and Tota, B.(2005). The heart of Sparus auratus: a reappraisal of cardiac functional morphology in teleosts. J. Exp. Zool. A303, 665-675.
Imbrogno, S., De Iuri, L., Mazza, R. and Tota, B.(2001). Nitric oxide modulates cardiac performance in the heart of Anguilla anguilla. J. Exp. Biol.204, 1719-1727.
Imbrogno, S., Cerra, M. C. and Tota, B.(2003). Angiotensin II-induced inotropism requires an endocardial endothelium-nitric oxide mechanism in the in-vitro heart of Anguilla anguilla. J. Exp. Biol.206, 2675-2684.
Imbrogno, S., Angelone, T., Corti, A., Adamo, C., Helle, K. B. and Tota, B.(2004). Influence of vasostatins, the chromogranin A-derived peptides, on the working heart of the eel (Anguilla anguilla): negative inotropy and mechanism of action. Gen. Comp. Endocrinol.139, 20-28.
Imbrogno, S., Angelone, T., Adamo, C., Pulerà, E., Tota, B. and Cerra, M. C.
(2006). Beta3-adrenoceptor in the eel (Anguilla anguilla) heart: negative inotropy and NO-cGMP-dependent mechanism. J. Exp. Biol.209, 4966-4973.
Imbrogno, S., Garofalo, F., Cerra, M. C., Mahata, S. K. and Tota, B.(2010). The catecholamine release-inhibitory peptide catestatin (chromogranin A344-363) modulates myocardial function in fish. J. Exp. Biol.213, 3636-3643.
Imbrogno, S., Tota, B. and Gattuso, A.(2011). The evolutionary functions of cardiac NOS/NO in vertebrates tracked by fish and amphibian paradigms. Nitric Oxide25, 1-10.
Iversen, N. K., McKenzie, D. J., Malte, H. and Wang, T.(2010). Reflex bradycardia does not influence oxygen consumption during hypoxia in the European eel (Anguilla anguilla). J. Comp. Physiol. B180, 495-502.
Iwakiri, Y., Satoh, A., Chatterjee, S., Toomre, D. K., Chalouni, C. M., Fulton, D., Groszmann, R. J., Shah, V. H. and Sessa, W. C.(2006). Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking. Proc. Natl. Acad. Sci. USA103, 19777-19782.
Jensen, F. B.(2009). The role of nitrite in nitric oxide homeostasis: a comparative perspective. Biochim. Biophys. Acta1787, 841-848.
Jensen, F. B. and Hansen, M. N.(2011). Differential uptake and metabolism of nitrite in normoxic and hypoxic goldfish. Aquat. Toxicol.101, 318-325.
Johnson, K. R. and Olson, K. R.(2009). The response of non-traditional natriuretic peptide production sites to salt and water manipulations in the rainbow trout. J. Exp. Biol.212, 2991-2997.
Johnson, R. K., Hering, D., Furse, M. T. and Verdonschot, P. F. M.(2006). Indicators of ecological change: comparison of the early response of four organism groups to stress gradients. Hydrobiologia566, 139-152.
Kaiya, H. and Takei, Y.(1996a). Changes in plasma atrial and ventricular natriuretic peptide concentrations after transfer of eels from freshwater to seawater or vice versa. Gen. Comp. Endocrinol.104, 337-345.
