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Morpho-functional characterization of the goldfish (Carassius auratus L.) heart
Comparative Biochemistry and Physiology, Part A 163 (2012) 215–222
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Morpho-functional characterization of the goldfish (Carassius auratus L.) heart F. Garofalo a, 1, S. Imbrogno a, 1, B. Tota a, D. Amelio a, b,⁎ a b
Department of Cell Biology, University of Calabria, Italy Department of Pharmaco-Biology, University of Calabria, Italy
a r t i c l e
i n f o
Article history: Received 23 April 2012 Received in revised form 31 May 2012 Accepted 31 May 2012 Available online 13 June 2012 Keywords: Fish heart Goldfish eNOS Heart morphology Cardiac performance
a b s t r a c t Using morphological and physiological approaches we provided, for the first time, a structural and functional characterization of Carassius auratus L. heart. Besides to the classical four chambers, i.e. sinus venosus, atrium, ventricle, bulbus, we described two distinct structures corresponding to the atrio-ventricular (AV) region and the conus arteriosus. The atrium is very large and highly trabeculated; the ventricle shows an outer compacta, vascularized by coronary vessels, and an inner spongiosa; the bulbus wall is characterized by a high elastin/ collagen ratio, which makes it extremely compliant. Immunolocalization revealed a strong expression of activated “eNOS-like” isoforms both at coronary endothelium and, to a lesser extent, in the myocardiocytes and the endocardial endothelium (EE). The structural design of the heart appears to comply with its mechanical function. Using an in vitro working heart preparation, cardiac performance was evaluated at different filling and afterload pressures. The hearts were very sensitive to filling pressure increases. Maximum Stroke volume (SV = 1.08 ± 0.09 mL/kg body mass) was obtained with an input pressure of 0.4 kPa. The heart was not able to sustain afterload increases, values higher than 1.5 kPa impairing its performance. These morpho-functional features are consistent with a volume pump mechanical performance. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The fish heart exhibits an impressive morpho-functional flexibility in relation to both developmental and ecophysiological changes. This flexibility, clearly exemplified by the relationships between the myoarchitecture of the ventricular pump and its mechanical performance, is an issue of elevated interest in comparative cardiac morphodynamics (Tota and Gattuso, 1996; Cerra et al., 2004). A notable aspect of this cardiac flexibility is highlighted by the relationship between the structural organization of the ventricular pump and the mechanical performance of the heart, evaluated in terms of the relative contribution of pressure and volume work to the stroke work (Tota and Gattuso, 1996). This provides an insight into how the internal construction of the ventricular chamber is adapted to its functional performance. Numerous studies have reviewed the structural organization of the fish heart chambers also in terms of cardiac performance (Santer, 1985; Satchell, 1991; Farrell and Jones, 1992; Burggren et al., 1997). On the basis of the external shape, fish heart ventricle has been classified into three major categories, i.e. sac-like, which appears rounded and with a blunt apex; tubular, showing a cylindrical cross section; and pyramidal, with a triangular base (Santer et al., 1983; Santer, 1985, and references therein).
⁎ Corresponding author at: Department of Cell Biology and Pharmaco-Biology, University of Calabria, 87030 Arcavacata di Rende, CS, Italy. Tel.: + 39 0984 492909; fax: + 39 0984 492906. E-mail address: [email protected] (D. Amelio). 1 These authors equally contributed to the work. 1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2012.05.206
Moreover, the different myocardial arrangement allowed to distinguish four different ventricle type (Tota et al., 1983; Tota, 1989; Farrell and Jones, 1992 and references therein). In the Type I the ventricular myocardium appears completely avascular and trabeculated. The Type II presents both an external compacta (vascularized) layer and an inner spongiosa. Ventricles of type III show vessels both in the compacta and spongiosa. Finally, the Type IV includes ventricles prevalently formed by compact vascularized myocardium. Fish of the genus Carassius (Cypriniformes, Cyprinidae) represent advanced forms of teleosts largely used as model organisms in the fields of molecular evolution and comparative genomics (Luo et al., 2006), cell biology (Lee et al., 1997), immunology (Hanington et al., 2006) and neurobiology (Huesa et al., 2005; Preuss et al., 2006). Both the goldfish (Carassius auratus) and the crucian carp (Carassius carassius) are able to tolerate prolonged and severe hypoxic conditions and remain active when overwintering in ice-covered ponds (Bickler and Buck, 2007). Obviously, this requires the molecular machinery which sustains myocardial contractility to preserve its function. Also several reptiles, such as the turtles, are surprising in their ability to face acidosis which follows the hypoxic/anoxic conditions. Regardless the similar response, each species faces acidosis by activating different strategies. For example, in the crucian carp and the goldfish, the lactic acidosis which follows the hypoxic/anoxic conditions is prevented by converting lactate to ethanol and CO2, both excreted by the gills (Bickler and Buck, 2007). In this context, the teleost C. auratus has been so far regarded as a precious model to study the mechanisms which allow survival and heart function preservation
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when O2 availability becomes a limiting factor (Pedersen et al., 2010 and references therein). However, despite the numerous studies on the physiology and behavior of the goldfish and its cardiac tolerance to anoxia, the basal morphological and functional traits of its heart have not yet been analyzed. To fill this gap is a fundamental prerequisite in order to explore the molecular mechanisms of cardiac plasticity, as well as their evolutionary history. In this study we used both light and electron microscopy to provide a morphological characterization of C. auratus heart. Moreover, by physiological experiments performed on the isolated and perfused working heart we described the mechanical behavior of the intact organ. The morpho-functional features well comply with the goldfish heart as a volume pump, i.e. it is able to produce mainly volume work. 2. Materials and methods 2.1. Animals Specimens of goldfish C. auratus (n = 40; mass: 63.8 ± 19.8 g, mean±SD), provided by a fish farm (COF SAS, Bologna, Italy), were kept at room temperature (18–20 °C) for 7–10 days. They were anesthetized with a dose of MS222 (tricaine methane sulfonate, Sigma-Aldrich Chemical Co., UK). In accordance with the accepted standards of animal care, the experiments were organized to minimize stress and number of animal used. 2.2. Morphological experiments The hearts, removed from the pericardial cavity and flushed with phosphate-buffered saline (PBS; pH: 7.6), were blocked in diastole with an excess of KCl (0.5 g/L) and processed according to the following procedures: 2.3. Scanning electron microscopy (SEM) Three hearts, dissected longitudinally under a stereomicroscope to expose the conus valves, were fixed in 3% glutaraldehyde in PBS for 3 h. They were then dehydrated in graded acetone, dried by the critical point method, and gold-sputter-coated. Observations were made using a Philips SEM 501 scanning microscope. 2.4. Light microscopy The hearts of 4 specimens were fixed in MAW fixative (methanol– acetone–water, 2:2:1), dehydrated in graded ethanol, embedded in paraplast (Sherwood, St. Louis, MO, USA), and serially sectioned at 8 μm. The sections were placed onto Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). Several sections were stained with either hematoxylin and eosin for a general assessment of tissue structure, Gomori's Trichrome for connective tissue, orcein for detection of elastic fibers and Sirius red for detection of collagen fibers. In addition, three hearts were fixed in 3% glutaraldehyde in PBS for 3 h. Fragments of the ventricle were postfixed in 1% osmium tetroxide, dehydrated in graded acetone and propylene oxide, and embedded in Araldite (Fluka). Semithin (1 μm) sections were cut with a Leica ultracut UCT, stained with 1% toluidine blue, and observed with a Zeiss III photomicroscope. 2.5. Immunofluorescence For phosphorylated (Ser 1177) eNOS immunodetection, sections, obtained as above mentioned, were deparaffined, rehydrated, rinsed in TBS and incubated with 1.5% BSA in TBS for 1 h. Subsequently they were incubated overnight at 4 °C with polyclonal anti p-eNOS (Ser 1177) antibody (Santa Cruz Biotechnology; 1:100), developed in goat against a short amino acid sequence containing phosphorylated
Ser 1177 of eNOS of human origin. To detect the p-eNOS signal, after washing in TBS (3× 10 min) the slides were incubated with FITCconjugated anti goat IgG (Sigma-Aldrich, St. Louis, MO, USA; 1:100). Slides were then mounted with mounting medium (Vectashield, Vector Laboratories) and observed under a microscope (Leica DMI 4000 B). Negative controls were obtained on parallel sections treated in the same manner, excluding primary antibody. For nuclear counterstaining, sections were incubated with Hoechst (Sigma, USA; 1:1000) for 5 min. 2.6. Physiological experiments 2.6.1. Isolated and perfused in vitro working heart preparation The hearts, removed without the parietal pericardium and cannulated, were transferred to a perfusion chamber filled with Ringer's solution and connected to a perfusion apparatus as described by Tota et al. (1991). The hearts received Ringer's solution from an input reservoir and pumped against an afterload pressure given by the height of an output reservoir. Ringer's solution was (in mmol/L) NaCl 124.9, KCl 2.49, MgSO4 0.94, NaH2PO4 1, Glucose 5, NaHCO3 15 and CaCl2 1.2; it was equilibrated with a mixture of O2:CO2 at 99.5%:0.5%. Experiments were carried out at room temperature (18–20 °C). Hemodynamic parameters were measured through two MP-20D pressure transducers (Micron Instruments, Simi Valley, CA, USA) connected to a PowerLab data acquisition system and analyzed by using Chart software (ADInstruments, Basile, Italy). Pressures were corrected for cannula resistance. Heart rate (HR) was calculated from pressure recording curves. Cardiac output (CO), was collected over 1 min and weighed; values were corrected for fluid density and expressed as volume measurements. Stroke volume (SV, CO/HR) was used as a measure of ventricular performance. 2.7. Experimental protocols 2.7.1. Basal conditions Basal conditions were established by evaluating cardiac performance at different filling and afterload pressures. The heart generated its own rhythm. Cardiac variables were measured simultaneously during experiments. Hearts that did not stabilize within 20 min of perfusion were discarded. 2.7.2. Time-course experiments To assess the endurance of the preparation and to detect the onset of the hypodynamic state, the cardiac performance variables under basal conditions were measured every 10 min of perfusion for approximately 160 min. 2.7.3. Frank–Starling response To evaluate the Frank–Starling response of the heart, after the stabilization period (15–20 min), starting from basal conditions, filling pressure was increased until there was no further discernible increase in CO. For each filling pressure increase, the variables of cardiac performance were measured after a 5 min perfusion with saline. Each increment was 0.5 cmH2O. The output pressure was stable at 1.5 kPa. 3. Results 3.1. Morphological results As shown in Fig. 1, the heart of C. auratus appears constituted by the typical four chambers i.e. sinus venosus, atrium, ventricle and bulbus arteriosus. In addition, an AV region and a muscularized conus arteriosus supporting the conus valves can be recognized. 3.1.1. Sinus venosus and atrium The sinus venosus, which directs the blood into the atrial cavity, is mostly made up of connective tissue (Fig. 2A). In proximity of the
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Fig. 1. (A) C. auratus heart after M.A.W. fixation (dorsal right view). (B) Sagittal section of the heart hematoxilin–eosin stained. v = ventricle; a = atrium; b = bulbus; red arrows and sv = sinus venosus; av = atrio-ventricular region; ca = conus arteriosus.
sino-atrial region, rings of nervous tissue resembling “ganglion cells” are evident (Fig. 2A). The single atrium chamber is remarkably large with a size comparable to that of the ventricle (Fig. 1A,B); the mean percentage of atrial mass with respect to the ventricular and body mass resulted in 27.6% and 0.021%, respectively. Histological sections of the atrium stained with hematoxilin/eosin show the presence of a complex network of thin trabeculae that divide the atrial cavity into lacunae, progressively smaller moving from the lumen toward the epicardium (Fig. 1B). As shown in the sections stained with Sirius red, the myocardial tissue is surrounded by a subepicardial layer of collagen fibers, also evident around the trabeculae at subendocardial level (Fig. 2B). In orcein stained atrial sections, elastin showed a distribution pattern similar to that of collagen (data not shown).
The atrio-ventricular (AV) region provides a support for the AV valves consisting of a ring of compacta which appears vascularized (Fig. 2C) and surrounded by connective tissue formed by loose collagen fibers delimitating the AV region (Fig. 2D,E). The AV valves are formed by two leaflets, which arise from the AV muscle ring. The leaflets contain numerous cells grouped into a dense core and variable amounts of collagen (Fig. 2D) and elastin (Fig. 2E), particularly abundant at the level of the thick atrial fibrosa (Fig. 2D,E). 3.1.2. Ventricle The ventricle shows a sac-like shape (Fig. 1A) and is made up of an outer compact, i.e. compacta, and an inner trabeculate, i.e. spongiosa, layer (Figs. 2F; 3A–E). The compacta is formed by bundle of muscle
Fig. 2. (A) Sinus venosus (sv) wall containing rings (red arrows) of nervous tissue corresponding to the primitive pacemaker region. Sagittal section of atrium stained with Sirius red (B). Longitudinal section of the atrio-ventricular region stained with Sirius red (C,D) and orcein (E). In C, a detail of vessels (green arrows) in the muscular tissue supporting the atrio-ventricular valves is shown. In D, the connective ring (black arrows) surrounding the vascularized muscular tissue (blue arrows) is evident. Note the thick fibrosa of the atrio-ventricular valves containing a large amount of collagen (D; red arrows) and elastin (E; pink arrows). (F) Sagittal section of ventricle stained with Gomori's Trichrome; the border between compacta (c) and spongiosa (s) layers is evident (black arrows). Ep = epicardium; a = atrium; v = ventricle; c = compacta; s = spongiosa.
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Fig. 3. SEM micrographs illustrating the anatomy of the C. auratus heart (A,B,C). Details of compacta and spongiosa are evident in D,E, and F. The conus arteriosus and the conus valves are shown in C. va = ventral aorta; b = bulbus; a = atrium; v = ventricle; c = compacta; s = spongiosa; ca = conus arteriosus; asterisks = conus valves. In D and E, compacta (red arrows) and subepicardial (white arrow) vessels are indicated. In F, note the endocardial bridges (yellow arrows).
tissue variously oriented and vascularized by coronary vessels (Figs. 2F; 3D,E). The compacta appears thicker at the base of the ventricle and thinner toward the apex (Figs. 1B; 3B). A layer of collagen at the boundary between compacta and spongiosa is evident in the sections stained with Gomori's Trichrome (Fig. 2F). In contrast to the compacta, the spongiosa is avascular and contains numerous trabeculae that divide the lumen of the ventricle into small lacunae. The intertrabecular spaces are progressively smaller moving from the lumen toward the epicardium (Figs. 1B; 3B). The trabeculae are covered by a thin layer of endocardial endothelium (EE) cells and are connected by “endocardial bridges” (Fig. 3F). 3.1.3. Conus arteriosus An anatomical segment, corresponding to the conus arteriosus, is interposed between the ventricle and the bulbus, providing support to the valve complex (Fig. 4A,B,C). The conus appears as a rim of myocardium located at the base of the ventricle (Figs. 4A,B,C and 3C), constituted by compact vascularized (Fig. 4C) myocardium containing a large amount of elastin and collagen fibers. Collagen and loose connective tissue separate the conus arteriosus from the ventricular myocardium and the bulbus, respectively (Fig. 4A,B,C). The valve complex consists of two conus valves (left and right), each formed by two components, the leaflet and its supporting structure, the sinus (Figs. 3C; 4A,B,C). Each leaflet presents a corpulent proximal body, anchored to the conus, and a distal region oriented toward the bulbus (Fig. 4A,B). On the ventricular side, the leaflets present a thick fibrosa rich both in elastin (Fig. 4A) and collagen (Fig. 4B,C). The sinus wall is rich in elastin (Fig. 4A) and collagen (Fig. 4B,C) and thickens toward the bulbus. As firstly highlighted in various teleosts by Icardo and coworkers (2005), the sinus represents the anatomical linkage between three different structures: ventricle, conus arteriosus and bulbus. In fact, its external layer is attached to the conus myocardium (in the proximal portion),
the loose subepicardial tissue (in the middle portion) and the inner surface of the bulbus (in the distal portion) (Fig. 4A,B,C). 3.1.4. Bulbus arteriosus The wall of the bulbus is organized into layers: the endocardium, the endocardial ridges, the middle layer, the subepicardium and the epicardium. The columnar ridges (Figs. 1B, 3A,C and 4D,E), occupying almost the whole area of lumen are covered by endocardial cells and appear thicker at the base of the bulbus and thinner toward the ventral aorta (Figs. 1B and 4E). The middle layer contains loose smooth muscle cells interposed between a large amount of elastin (Fig. 4E) mixed with a small amount of collagen fibers (Fig. 4D). On the contrary, the subepicardial layer appears rich in collagen fibers and vessels (Fig. 4D). The bulbus is in continuity with the ventral aorta which presents a thick elastic wall (Fig. 4F), containing collagen fibers prevalently at the sub-endothelial and at the external layer of the tunica media (Fig. 4G). On the adventitia of the ventral aorta, a distinct vessel, belonging to the hypobranchial arterial system and surrounded by loose connective tissue, is evident (Fig. 4F,G). 3.2. eNOS immunodetection The immunolocalization of a phosphorylated eNOS-like isoform (for terminology and references in fish, see Imbrogno et al., 2011), obtained by using polyclonal anti p-eNOS (ser 1177) specific antibody, is shown in Fig. 5A,B. The enzyme is localized in the atrial (data not shown) and in the ventricular myocardium (Fig. 5A,B) of the goldfish heart. Labeling specificity was confirmed by the absence of the signal in parallel control sections treated without the primary antibody (Fig. 5C). In particular, p-eNOS was prevalently localized at the level of the vascular endothelium and, to a lesser extent, in the myocardiocytes and the endocardial endothelium (EE) (Fig. 5A,B).
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Fig. 4. Orcein (A) and Sirius red (B) staining of conus arteriosus. The thick fibrosa (ventricular layer of the conus valvar leaflets) rich in elastin (red arrows) and collagen (green arrows) is evident. (C) Detailed view of conus arteriosus; note the vessels (orange arrows) in the compact myocardium. Sirius red (D,G) and orcein (E,F) staining of bulbus longitudinal sections. In F and G, a transversal cut of the ventral aorta and the hypobranchial artery (black arrows) is evident. ca= conus arteriosus; sw = sinus wall; b = bulbus; v = ventricle; va= ventral aorta; Ep= epicardium; Sep = subepicardium; l = lumen.
3.3. Physiological results 3.3.1. Isolated working heart preparations By evaluating cardiac performance at different filling and afterload pressures, basal performance parameters were set up to a mean output pressure of approximately 1.5 kPa and cardiac output of about 10–12 mL/min/kg body mass. The basal hemodynamic parameters of isolated and perfused heart of C. auratus are reported in Table 1. Typical time-course curves of SV and HR indicated that the performance of the heart was stable for more than 2 h, after which the heart fell into a hypodynamic state characterized by a linear decrease in SV (data not shown). 3.3.2. Frank–Starling response The perfused C. auratus heart showed a typical Frank–Starling response. Increases of filling pressure (from 0.1 to 0.4 kPa) produced corresponding increases of SV. The maximum SV = 1.08 ± 0.09 mL/kg body mass was obtained with an input pressure of 0.4 kPa. Notably, we observed that the heart was not able to sustain afterload increases, as documented by the finding that values higher than 1.5 kPa resulted in a rapid decrease of cardiac performance (data not shown). 4. Discussion Although the evolution of the fish heart shows a high level of genetic conservation, the use of the same basic building blocks during its developmental processes has resulted in a remarkable variety of cardiac
designs (Olson, 2006). To some extent, such variety mirrors the impressive morpho-functional flexibility of the piscine cardiovascular system in relation to developmental and often extreme eco-physiological challenges associated with body growth and life style (Tota and Gattuso, 1996). Here we provide a morpho-functional characterization of the C. auratus four-chambered heart, i.e., sinus venosus, atrium, ventricle and bulbus, including the two distinct structures corresponding to the AV region and the conus arteriosus. The sinus venosus, which receives the peripheral venous blood to be ejected into the atrium, is made up of a thin wall and shows, in the proximity of the sino-atrial region, rings of nervous tissue, organized in cluster of ganglion cells probably corresponding to the primitive pacemaker region described in other teleosts (Yamauchi, 1980). In fishes, the atrium presents species-specific differences in both size and shape (Farrell and Jones, 1992). In C. auratus, the atrium appears as a large chamber completely trabeculated and rich in collagen and elastin. As extensively demonstrated by Icardo and Colvee (2011), the AV region, i.e. the area of the heart connecting the atrium and the ventricle, represents a distinct species-specific segment of the teleost heart, exhibiting relevant evolutionary diversification. The abundance of a vascularized compact myocardium in the C. auratus AV region represents a feature common to different teleosts with a Type II ventricle (Icardo and Colvee, 2011). In addition, as in other teleost species, also in C. auratus this region is surrounded by a ring of connective tissue formed by loose collagen fibers. According to the goldfish ventricle myoarchitecture and blood supply, the presence of both a vascularized compacta and a trabeculated
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Fig. 5. p-eNOS immunolocalization in the C. auratus ventricle (A,B). A strong signal appears on the vascular endothelium (yellow arrows). A weak signal is confined to the endocardial endothelium (red arrows) and in the myocardiocytes (pink arrows). Negative control is shown in C. In A and C nuclei are counterstained with Hoechst.
spongiosa allows to classify it as a Type II heart (Tota et al., 1983; Tota, 1989). The particular location of collagen fibers at the boundary between compacta and spongiosa suggests their possible role as a bonding glue to mechanically link the two differently oriented myocardial layers (Poupa et al., 1974; Tota, 1978; Icardo et al., 2005). In fact, while in the compacta the cardiomyocytes are oriented circumferentially, in the spongiosa they show a prevalent perpendicular trend. Conversely, in the salmonid heart, Pieperhoff et al. (2009) showed the absence of a continuous layer of collagen fibers and high amount of desmosomelike and fascia adherens-like structures that could function as anchorage structures between the compacta and the spongiosa. On the basis of these observations, it is conceivable that many structural features may concur to provide a morpho-functional boundary between the two muscular layers. Collagen fibers are also evident in the subepicardial layer where they can limit the extreme expansion of the ventricular chamber increasing its resilience (Icardo et al., 2005). Recent studies (Icardo et al., 2003; Icardo, 2006) demonstrated both in modern and in ancient teleosts the presence of a muscularized and vascularized conus arteriosus, interposed between the ventricle and the bulbus. We confirm its occurrence also in the goldfish heart where the conus, offering support to the valve complex, appears as a crown of tissue located at the base of the ventricle. The sinus valve appears as an element of continuity between ventricle, conus and bulbus. Moreover, for its abundance in elastin, which suggests a high deformability during cardiac cycle, it could play a role in facilitating heart mechanics. In fact, the high elasticity supports both the deformation during the dilation and the subsequent recovery of bulbus arteriosus; at the same time, by decreasing the conus stress, it favors valvar closure. In addition, the large amount of collagen and elastin of the thick ventricular fibrosa of the valve leaflets might exert a local resistance to the hemodynamic force applied on this side during the ventricular systole. The robust bulbus chamber contains a large amount of elastin, similar to that reported in carp (90%: Licht and Harris, 1973), which makes this cardiac chamber extremely flexible. In fish, the elevated compliance of the bulbus wall has been related to the high elastin/ collagen ratio (see for example the trout: Serafini-Fracassini et al., 1978). The gradual elastic recovery of the bulbus (windkessel effect: Farrell and Jones, 1992, and references therein), well evidenced by our functional experiments, ensures a constant blood flow towards the gills, preventing the damage of the delicate branchial vasculature as illustrated by Jones et al. (1993) in other teleosts. At the same time, the control of an excessive bulbus expansion during ventricular
systole may be guaranteed by high levels of collagen fibers, particularly localized in the subepicardium. The relationships between the structural design of the ventricular pump and its morphodynamic behavior have been largely investigated both in mammalian and non-mammalian vertebrates. In various fish species, this relationship, evaluated in terms of the relative contribution of pressure and volume work to the stroke work, allows a distinction between ventricles producing mainly volume work and those producing mainly pressure work (Tota and Gattuso, 1996; Icardo et al., 2005). C. auratus heart appears to exhibit a volume pump mechanical behavior. In fact, although under basal conditions (see Table 1), the CO resulted in 11.85 mL/min/kg, a value similar to those reported for other teleosts (Farrell and Jones, 1992), including the crucian carp (8.4 mL/min/kg at 8 °C; Farrell and Stecyk, 2007), the striking sensitivity to filling pressure changes, documented by the elevated SV values raised in response to increasing filling pressure, clearly indicates a hemodynamic ability to displace high blood volumes at low pressures. In this context, the elevated atrial/ventricular relative mass (27,6%), higher than that observed in other teleosts (e.g. eel: 18.4%, unpublished data), as well as the presence of large lacunary spaces, well comply with the mechanical behavior of the goldfish heart as volume pump. On the other hand, a typical volume pump heart is suited for maintaining constant the stroke volume only within a narrow range of afterloads. In fact, as highlighted by the Antarctic icefish paradigm (in vivo: Hemmingsen et al., 1972; in vitro: Tota et al., 1991), the output pressure values above which both cardiac work and power generation are quickly impaired result very low. Of note, in the in vivo crucian carp (C. carassius), which retains normal cardiac performance and autonomic cardiovascular regulation for at least 5 days of anoxia, the ventral aortic pressure is about 1.5 kPa (Stecyk et al., 2004). Interestingly, similarly to its relative crucian carp, the goldfish heart works at a very low output pressure and, in contrasts with several temperate teleosts able to sustain high values of afterloads [up to 5 kPa (Farrell and Jones, 1992; Tota and Gattuso, 1996)], showed an inability to maintain a constant systolic function in response to afterload increases. In turn, the latter caused an evident distension of the bulbus chamber, likely related to its high elastin/ collagen ratio. Whether the emptying constraint of the bulbus could be due to a particular fibrillar design of its wall remains to be clarified (for other teleost species see Braun et al., 2003). This behavior of C. auratus heart as volume pump is well evident when analyzed in the comparative context of the mechanical performance of
Table 1 Performance variables under basal conditions of isolated C. auratus heart preparations perfused with oxygen saturated Ringer's solution (n = 25). Heart rate beats min− 1
Filling pressure kPa
Output pressure kPa
Cardiac output ml min− 1 kg− 1
Stroke volume ml kg− 1
Power output mW g− 1
56.21 ± 3.24
0.11 ± 0.03
1.51 ± 0.05
11.85 ± 0.31
0.2 ± 0.02
0.36 ± 0.01
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Fig. 6. Values for afterload and stroke volume in elasmobranchs and teleosts under basal conditions. Bars represent the increases of afterload and stroke volume in conditions of maximum sustainable output or input pressures, respectively (modified from Tota and Gattuso, 1996).
different hearts, expressed in terms of pressure generation and volume movement, as illustrated in Fig. 6. In the fish heart, a widespread expression of a constitutive eNOSlike isoform, at vascular, endocardium endothelium and myocardiocytes levels, has been documented. Together with parallel physiopharmacological data, this points to a major role of the NOS/NO system in the fish heart (Imbrogno et al., 2011, and references therein). Consistent with our immunological findings, a key role of the NOS/NO system in the modulation of cardiac function in the goldfish heart may be hypothesized. Recent studies have highlighted the importance of the intracellular NO homeostasis during hypoxia in C. auratus heart (Hansen and Jensen, 2010; Pedersen et al., 2010; Jensen and Hansen, 2011). In particular, it has been supposed that when the NOS-mediated NO formation is compromised by a shortage of the substrate O2, an increase of tissue NOS expression to keep cellular NO levels stable may be expected (Hansen and Jensen, 2010). Studies are underway in our laboratory to clarify the putative cardiomodulatory role of the NOS/NO system as well as the existence of oxygen availability-related differences in the eNOS-like expression and localization in the goldfish heart. The present detection of intracardiac NOS in goldfish represents a prerequisite for this purpose. In conclusion, the striking goldfish tolerance against prolonged and severe hypoxia requires a balance between energy supply and demand, as well as coping with the acidosis associated with anaerobic end-product accumulation, i.e. lactic acidosis. This is prevented by goldfish through lactate conversion to ethanol and CO2, both excreted by the gills (Shoubridge and Hochachka, 1980; Bickler and Buck, 2007). Conceivably, from a morpho-functional standpoint, the volume pump ability of the goldfish heart to generate elevated CO and to displace in the branchial circulation a relatively large blood volume at low pressure may benefit the ethanol shuttling to the gills, thereby preventing its accumulation and even tissue intoxication. Moreover, the low values of PO, very similar to that estimated for the crucian carp in vivo (Farrell and Stecyk, 2007) and enough to be supported by anaerobic glycolysis, may ensure the goldfish heart to continue to beat during anoxic conditions. Acknowledgments This work was supported by “Ministero dell'Istruzione, dell'Università e della Ricerca” (e.g. MURST 60%) and by the Italian National Research Program in Antarctica (PNRA). The authors would like to thank Dr. E. Perrotta for electronmicroscopy technical assistance.
References Bickler, P.E., Buck, L.T., 2007. Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Annu. Rev. Physiol. 69, 145–170. Braun, M.H., Brill, R.W., Gosline, J.M., Jones, D.R., 2003. Form and function of the bulbus arteriosus in yellowfin tuna (Thunnus albacares), bigeye tuna (Thunnus obesus) and blue marlin (Makaira nigricans): static properties. J. Exp. Biol. 206, 3311–3326. Burggren, W.W., Farrell, A.P., Lillywhite, H., 1997. Vertebrate cardiovascular systems. In: Dantzler, W.H. (Ed.), Handbook of Physiology, sect. 13, Comparative Physiology, vol. 1. Oxford University Press, New York, pp. 215–308. Cerra, M.C., Imbrogno, S., Amelio, D., Garofalo, F., Colvee, E., Tota, B., Icardo, J.M., 2004. Cardiac morphodynamic remodeling in the growing eel. J. Exp. Biol. 207, 2867–2875. Farrell, A.P., Jones, D.R., 1992. The heart. Fish Physiol. XII, Part A, 1–73. Farrell, A.P., Stecyk, J.A.W., 2007. The heart as a working model to explore themes and strategies for anoxic survival in ectothermic vertebrates. Comp. Biochem. Physiol. A 147, 300–312. Hanington, P.C., Barreda, D.R., Belosevic, M., 2006. A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages. J. Biol. Chem. 281, 9963–9970. Hansen, M.N., Jensen, F.B., 2010. Nitric oxide metabolites in goldfish under normoxic and hypoxic conditions. J. Exp. Biol. 213, 3593–3602. Hemmingsen, E.A., Douglas, E.L., Johansen, K., Millard, R.W., 1972. Aortic blood flow and cardiac output in the hemoglobin-free fish Chaenocephalus aceratus. Comp. Biochem. Physiol. A 43, 1045–1051. Huesa, G., van den Pol, A.N., Finger, T.E., 2005. Differential distribution of hypocretin (orexin) and melanin-concentrating hormone in the goldfish brain. J. Comp. Neurol. 488, 476–491. Icardo, J.M., 2006. Conus arteriosus of the teleost heart: dismissed, but not missed. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 288, 900–908. Icardo, J.M., Colvee, E., 2011. The atrioventricular region of the teleost heart. A distinct heart segment. Anat. Rec. 294, 236–242. Icardo, J.M., Schib, J.L., Ojeda, J.L., Duràn, A.C., Guerrero, A., Colvee, E., Amelio, D., Sans-Coma, V., 2003. The conus valves of the adult gilthead seabream (Sparus auratus). J. Anat. 202, 537–550. Icardo, J.M., Imbrogno, S., Gattuso, A., Colvee, E., Tota, B., 2005. The heart of Sparus auratus: a reappraisal of cardiac functional morphology in teleosts. J. Exp. Zoolog. A Comp. Exp. Biol. 303, 665–675. Imbrogno, S., Tota, B., Gattuso, A., 2011. The evolutionary functions of cardiac NOS/NO in vertebrates tracked by fish and amphibian paradigms. Nitric Oxide 25, 1–10. Jensen, F.B., Hansen, M.N., 2011. Differential uptake and metabolism of nitrite in normoxic and hypoxic goldfish. Aquat. Toxicol. 101, 318–325. Jones, D.R., Brill, R.W., Bushnell, P.G., 1993. Ventricular and arterial dynamics of anaesthetised and swimming tuna. J. Exp. Biol. 182, 97–112. Lee, L.E., Caldwell, S.J., Gibbons, J., 1997. Development of a cell line from skin of goldfish, Carassius auratus, and effects of ascorbic acid on collagen deposition. Histochem. J. 29, 31–43. Licht, J.H., Harris, W.S., 1973. The structure, composition and elastic properties of the teleost bulbus arteriosus in the carp, Cyprinius carpio. Comp. Biochem. Physiol. A 46 699–670. Luo, J., Lang, M., Salzburger, W., Siegel, N., Stölting, K.N., Meyer, A., 2006. A BAC library for the goldfish Carassius auratus auratus (Cyprinidae, Cypriniformes). J. Exp. Zoolog. B 306, 567–574. Olson, E.N., 2006. Gene regulatory networks in the evolution and development of the heart. Science 313, 1922–1927. Pedersen, C.L., Faggiano, S., Helbo, S., Gesser, H., Fago, A., 2010. Roles of nitric oxide, nitrite and myoglobin on myocardial efficiency in trout (Oncorhynchus mykiss) and goldfish (Carassius auratus): implications for hypoxia tolerance. J. Exp. Biol. 213, 2755–2762.
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Pieperhoff, S., Bennett, W., Farrell, A.P., 2009. The intercellular organization of the two muscular systems in the adult salmonid heart, the compact and the spongy myocardium. J. Anat. 215 (5), 536–547. Poupa, O., Gesser, H., Jonsson, S., Sullivan, L., 1974. Coronary-supplied compact shell of ventricular myocardium in salmonids: growth and enzyme pattern. Comp. Biochem. Physiol. A 48, 85–95. Preuss, T., Osei-Bonsu, P.E., Weiss, S.A., Wang, C., Faber, D.S., 2006. Neural representation of object approach in a decision-making motor circuit. J. Neurosci. 26, 3454–3464. Santer, R.M., 1985. Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol. 89, 1–102. Santer, R.M., Greer Walker, M., Emerson, L., Witthames, P.R., 1983. On the morphology of the heart ventricle in marine teleost fist (teleostei). Comp. Biochem. Physiol. A 76, 453–457. Satchell, G.H., 1991. Physiology and Form of Fish Circulation. Cambridge University Press, Cambridge. Serafini-Fracassini, A., Field, J.M., Spina, J., Garbia, S., Stuart, R.J., 1978. The morphological organization and ultrastructure of elastin in the arterial wall of trout (Salmo gairdneri) and salmon (Salmo salar). J. Ultrastruct. Res. 65, 1–12. Shoubridge, E.A., Hochachka, P.W., 1980. Ethanol: novel end product of vertebrate anaerobic metabolism. Science 209, 308–309.
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Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Morpho-functional characterization of the goldfish (Carassius auratus L.) heart F. Garofalo a, 1, S. Imbrogno a, 1, B. Tota a, D. Amelio a, b,⁎ a b
Department of Cell Biology, University of Calabria, Italy Department of Pharmaco-Biology, University of Calabria, Italy
a r t i c l e
i n f o
Article history: Received 23 April 2012 Received in revised form 31 May 2012 Accepted 31 May 2012 Available online 13 June 2012 Keywords: Fish heart Goldfish eNOS Heart morphology Cardiac performance
a b s t r a c t Using morphological and physiological approaches we provided, for the first time, a structural and functional characterization of Carassius auratus L. heart. Besides to the classical four chambers, i.e. sinus venosus, atrium, ventricle, bulbus, we described two distinct structures corresponding to the atrio-ventricular (AV) region and the conus arteriosus. The atrium is very large and highly trabeculated; the ventricle shows an outer compacta, vascularized by coronary vessels, and an inner spongiosa; the bulbus wall is characterized by a high elastin/ collagen ratio, which makes it extremely compliant. Immunolocalization revealed a strong expression of activated “eNOS-like” isoforms both at coronary endothelium and, to a lesser extent, in the myocardiocytes and the endocardial endothelium (EE). The structural design of the heart appears to comply with its mechanical function. Using an in vitro working heart preparation, cardiac performance was evaluated at different filling and afterload pressures. The hearts were very sensitive to filling pressure increases. Maximum Stroke volume (SV = 1.08 ± 0.09 mL/kg body mass) was obtained with an input pressure of 0.4 kPa. The heart was not able to sustain afterload increases, values higher than 1.5 kPa impairing its performance. These morpho-functional features are consistent with a volume pump mechanical performance. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The fish heart exhibits an impressive morpho-functional flexibility in relation to both developmental and ecophysiological changes. This flexibility, clearly exemplified by the relationships between the myoarchitecture of the ventricular pump and its mechanical performance, is an issue of elevated interest in comparative cardiac morphodynamics (Tota and Gattuso, 1996; Cerra et al., 2004). A notable aspect of this cardiac flexibility is highlighted by the relationship between the structural organization of the ventricular pump and the mechanical performance of the heart, evaluated in terms of the relative contribution of pressure and volume work to the stroke work (Tota and Gattuso, 1996). This provides an insight into how the internal construction of the ventricular chamber is adapted to its functional performance. Numerous studies have reviewed the structural organization of the fish heart chambers also in terms of cardiac performance (Santer, 1985; Satchell, 1991; Farrell and Jones, 1992; Burggren et al., 1997). On the basis of the external shape, fish heart ventricle has been classified into three major categories, i.e. sac-like, which appears rounded and with a blunt apex; tubular, showing a cylindrical cross section; and pyramidal, with a triangular base (Santer et al., 1983; Santer, 1985, and references therein).
⁎ Corresponding author at: Department of Cell Biology and Pharmaco-Biology, University of Calabria, 87030 Arcavacata di Rende, CS, Italy. Tel.: + 39 0984 492909; fax: + 39 0984 492906. E-mail address: [email protected] (D. Amelio). 1 These authors equally contributed to the work. 1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2012.05.206
Moreover, the different myocardial arrangement allowed to distinguish four different ventricle type (Tota et al., 1983; Tota, 1989; Farrell and Jones, 1992 and references therein). In the Type I the ventricular myocardium appears completely avascular and trabeculated. The Type II presents both an external compacta (vascularized) layer and an inner spongiosa. Ventricles of type III show vessels both in the compacta and spongiosa. Finally, the Type IV includes ventricles prevalently formed by compact vascularized myocardium. Fish of the genus Carassius (Cypriniformes, Cyprinidae) represent advanced forms of teleosts largely used as model organisms in the fields of molecular evolution and comparative genomics (Luo et al., 2006), cell biology (Lee et al., 1997), immunology (Hanington et al., 2006) and neurobiology (Huesa et al., 2005; Preuss et al., 2006). Both the goldfish (Carassius auratus) and the crucian carp (Carassius carassius) are able to tolerate prolonged and severe hypoxic conditions and remain active when overwintering in ice-covered ponds (Bickler and Buck, 2007). Obviously, this requires the molecular machinery which sustains myocardial contractility to preserve its function. Also several reptiles, such as the turtles, are surprising in their ability to face acidosis which follows the hypoxic/anoxic conditions. Regardless the similar response, each species faces acidosis by activating different strategies. For example, in the crucian carp and the goldfish, the lactic acidosis which follows the hypoxic/anoxic conditions is prevented by converting lactate to ethanol and CO2, both excreted by the gills (Bickler and Buck, 2007). In this context, the teleost C. auratus has been so far regarded as a precious model to study the mechanisms which allow survival and heart function preservation
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when O2 availability becomes a limiting factor (Pedersen et al., 2010 and references therein). However, despite the numerous studies on the physiology and behavior of the goldfish and its cardiac tolerance to anoxia, the basal morphological and functional traits of its heart have not yet been analyzed. To fill this gap is a fundamental prerequisite in order to explore the molecular mechanisms of cardiac plasticity, as well as their evolutionary history. In this study we used both light and electron microscopy to provide a morphological characterization of C. auratus heart. Moreover, by physiological experiments performed on the isolated and perfused working heart we described the mechanical behavior of the intact organ. The morpho-functional features well comply with the goldfish heart as a volume pump, i.e. it is able to produce mainly volume work. 2. Materials and methods 2.1. Animals Specimens of goldfish C. auratus (n = 40; mass: 63.8 ± 19.8 g, mean±SD), provided by a fish farm (COF SAS, Bologna, Italy), were kept at room temperature (18–20 °C) for 7–10 days. They were anesthetized with a dose of MS222 (tricaine methane sulfonate, Sigma-Aldrich Chemical Co., UK). In accordance with the accepted standards of animal care, the experiments were organized to minimize stress and number of animal used. 2.2. Morphological experiments The hearts, removed from the pericardial cavity and flushed with phosphate-buffered saline (PBS; pH: 7.6), were blocked in diastole with an excess of KCl (0.5 g/L) and processed according to the following procedures: 2.3. Scanning electron microscopy (SEM) Three hearts, dissected longitudinally under a stereomicroscope to expose the conus valves, were fixed in 3% glutaraldehyde in PBS for 3 h. They were then dehydrated in graded acetone, dried by the critical point method, and gold-sputter-coated. Observations were made using a Philips SEM 501 scanning microscope. 2.4. Light microscopy The hearts of 4 specimens were fixed in MAW fixative (methanol– acetone–water, 2:2:1), dehydrated in graded ethanol, embedded in paraplast (Sherwood, St. Louis, MO, USA), and serially sectioned at 8 μm. The sections were placed onto Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). Several sections were stained with either hematoxylin and eosin for a general assessment of tissue structure, Gomori's Trichrome for connective tissue, orcein for detection of elastic fibers and Sirius red for detection of collagen fibers. In addition, three hearts were fixed in 3% glutaraldehyde in PBS for 3 h. Fragments of the ventricle were postfixed in 1% osmium tetroxide, dehydrated in graded acetone and propylene oxide, and embedded in Araldite (Fluka). Semithin (1 μm) sections were cut with a Leica ultracut UCT, stained with 1% toluidine blue, and observed with a Zeiss III photomicroscope. 2.5. Immunofluorescence For phosphorylated (Ser 1177) eNOS immunodetection, sections, obtained as above mentioned, were deparaffined, rehydrated, rinsed in TBS and incubated with 1.5% BSA in TBS for 1 h. Subsequently they were incubated overnight at 4 °C with polyclonal anti p-eNOS (Ser 1177) antibody (Santa Cruz Biotechnology; 1:100), developed in goat against a short amino acid sequence containing phosphorylated
Ser 1177 of eNOS of human origin. To detect the p-eNOS signal, after washing in TBS (3× 10 min) the slides were incubated with FITCconjugated anti goat IgG (Sigma-Aldrich, St. Louis, MO, USA; 1:100). Slides were then mounted with mounting medium (Vectashield, Vector Laboratories) and observed under a microscope (Leica DMI 4000 B). Negative controls were obtained on parallel sections treated in the same manner, excluding primary antibody. For nuclear counterstaining, sections were incubated with Hoechst (Sigma, USA; 1:1000) for 5 min. 2.6. Physiological experiments 2.6.1. Isolated and perfused in vitro working heart preparation The hearts, removed without the parietal pericardium and cannulated, were transferred to a perfusion chamber filled with Ringer's solution and connected to a perfusion apparatus as described by Tota et al. (1991). The hearts received Ringer's solution from an input reservoir and pumped against an afterload pressure given by the height of an output reservoir. Ringer's solution was (in mmol/L) NaCl 124.9, KCl 2.49, MgSO4 0.94, NaH2PO4 1, Glucose 5, NaHCO3 15 and CaCl2 1.2; it was equilibrated with a mixture of O2:CO2 at 99.5%:0.5%. Experiments were carried out at room temperature (18–20 °C). Hemodynamic parameters were measured through two MP-20D pressure transducers (Micron Instruments, Simi Valley, CA, USA) connected to a PowerLab data acquisition system and analyzed by using Chart software (ADInstruments, Basile, Italy). Pressures were corrected for cannula resistance. Heart rate (HR) was calculated from pressure recording curves. Cardiac output (CO), was collected over 1 min and weighed; values were corrected for fluid density and expressed as volume measurements. Stroke volume (SV, CO/HR) was used as a measure of ventricular performance. 2.7. Experimental protocols 2.7.1. Basal conditions Basal conditions were established by evaluating cardiac performance at different filling and afterload pressures. The heart generated its own rhythm. Cardiac variables were measured simultaneously during experiments. Hearts that did not stabilize within 20 min of perfusion were discarded. 2.7.2. Time-course experiments To assess the endurance of the preparation and to detect the onset of the hypodynamic state, the cardiac performance variables under basal conditions were measured every 10 min of perfusion for approximately 160 min. 2.7.3. Frank–Starling response To evaluate the Frank–Starling response of the heart, after the stabilization period (15–20 min), starting from basal conditions, filling pressure was increased until there was no further discernible increase in CO. For each filling pressure increase, the variables of cardiac performance were measured after a 5 min perfusion with saline. Each increment was 0.5 cmH2O. The output pressure was stable at 1.5 kPa. 3. Results 3.1. Morphological results As shown in Fig. 1, the heart of C. auratus appears constituted by the typical four chambers i.e. sinus venosus, atrium, ventricle and bulbus arteriosus. In addition, an AV region and a muscularized conus arteriosus supporting the conus valves can be recognized. 3.1.1. Sinus venosus and atrium The sinus venosus, which directs the blood into the atrial cavity, is mostly made up of connective tissue (Fig. 2A). In proximity of the
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Fig. 1. (A) C. auratus heart after M.A.W. fixation (dorsal right view). (B) Sagittal section of the heart hematoxilin–eosin stained. v = ventricle; a = atrium; b = bulbus; red arrows and sv = sinus venosus; av = atrio-ventricular region; ca = conus arteriosus.
sino-atrial region, rings of nervous tissue resembling “ganglion cells” are evident (Fig. 2A). The single atrium chamber is remarkably large with a size comparable to that of the ventricle (Fig. 1A,B); the mean percentage of atrial mass with respect to the ventricular and body mass resulted in 27.6% and 0.021%, respectively. Histological sections of the atrium stained with hematoxilin/eosin show the presence of a complex network of thin trabeculae that divide the atrial cavity into lacunae, progressively smaller moving from the lumen toward the epicardium (Fig. 1B). As shown in the sections stained with Sirius red, the myocardial tissue is surrounded by a subepicardial layer of collagen fibers, also evident around the trabeculae at subendocardial level (Fig. 2B). In orcein stained atrial sections, elastin showed a distribution pattern similar to that of collagen (data not shown).
The atrio-ventricular (AV) region provides a support for the AV valves consisting of a ring of compacta which appears vascularized (Fig. 2C) and surrounded by connective tissue formed by loose collagen fibers delimitating the AV region (Fig. 2D,E). The AV valves are formed by two leaflets, which arise from the AV muscle ring. The leaflets contain numerous cells grouped into a dense core and variable amounts of collagen (Fig. 2D) and elastin (Fig. 2E), particularly abundant at the level of the thick atrial fibrosa (Fig. 2D,E). 3.1.2. Ventricle The ventricle shows a sac-like shape (Fig. 1A) and is made up of an outer compact, i.e. compacta, and an inner trabeculate, i.e. spongiosa, layer (Figs. 2F; 3A–E). The compacta is formed by bundle of muscle
Fig. 2. (A) Sinus venosus (sv) wall containing rings (red arrows) of nervous tissue corresponding to the primitive pacemaker region. Sagittal section of atrium stained with Sirius red (B). Longitudinal section of the atrio-ventricular region stained with Sirius red (C,D) and orcein (E). In C, a detail of vessels (green arrows) in the muscular tissue supporting the atrio-ventricular valves is shown. In D, the connective ring (black arrows) surrounding the vascularized muscular tissue (blue arrows) is evident. Note the thick fibrosa of the atrio-ventricular valves containing a large amount of collagen (D; red arrows) and elastin (E; pink arrows). (F) Sagittal section of ventricle stained with Gomori's Trichrome; the border between compacta (c) and spongiosa (s) layers is evident (black arrows). Ep = epicardium; a = atrium; v = ventricle; c = compacta; s = spongiosa.
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Fig. 3. SEM micrographs illustrating the anatomy of the C. auratus heart (A,B,C). Details of compacta and spongiosa are evident in D,E, and F. The conus arteriosus and the conus valves are shown in C. va = ventral aorta; b = bulbus; a = atrium; v = ventricle; c = compacta; s = spongiosa; ca = conus arteriosus; asterisks = conus valves. In D and E, compacta (red arrows) and subepicardial (white arrow) vessels are indicated. In F, note the endocardial bridges (yellow arrows).
tissue variously oriented and vascularized by coronary vessels (Figs. 2F; 3D,E). The compacta appears thicker at the base of the ventricle and thinner toward the apex (Figs. 1B; 3B). A layer of collagen at the boundary between compacta and spongiosa is evident in the sections stained with Gomori's Trichrome (Fig. 2F). In contrast to the compacta, the spongiosa is avascular and contains numerous trabeculae that divide the lumen of the ventricle into small lacunae. The intertrabecular spaces are progressively smaller moving from the lumen toward the epicardium (Figs. 1B; 3B). The trabeculae are covered by a thin layer of endocardial endothelium (EE) cells and are connected by “endocardial bridges” (Fig. 3F). 3.1.3. Conus arteriosus An anatomical segment, corresponding to the conus arteriosus, is interposed between the ventricle and the bulbus, providing support to the valve complex (Fig. 4A,B,C). The conus appears as a rim of myocardium located at the base of the ventricle (Figs. 4A,B,C and 3C), constituted by compact vascularized (Fig. 4C) myocardium containing a large amount of elastin and collagen fibers. Collagen and loose connective tissue separate the conus arteriosus from the ventricular myocardium and the bulbus, respectively (Fig. 4A,B,C). The valve complex consists of two conus valves (left and right), each formed by two components, the leaflet and its supporting structure, the sinus (Figs. 3C; 4A,B,C). Each leaflet presents a corpulent proximal body, anchored to the conus, and a distal region oriented toward the bulbus (Fig. 4A,B). On the ventricular side, the leaflets present a thick fibrosa rich both in elastin (Fig. 4A) and collagen (Fig. 4B,C). The sinus wall is rich in elastin (Fig. 4A) and collagen (Fig. 4B,C) and thickens toward the bulbus. As firstly highlighted in various teleosts by Icardo and coworkers (2005), the sinus represents the anatomical linkage between three different structures: ventricle, conus arteriosus and bulbus. In fact, its external layer is attached to the conus myocardium (in the proximal portion),
the loose subepicardial tissue (in the middle portion) and the inner surface of the bulbus (in the distal portion) (Fig. 4A,B,C). 3.1.4. Bulbus arteriosus The wall of the bulbus is organized into layers: the endocardium, the endocardial ridges, the middle layer, the subepicardium and the epicardium. The columnar ridges (Figs. 1B, 3A,C and 4D,E), occupying almost the whole area of lumen are covered by endocardial cells and appear thicker at the base of the bulbus and thinner toward the ventral aorta (Figs. 1B and 4E). The middle layer contains loose smooth muscle cells interposed between a large amount of elastin (Fig. 4E) mixed with a small amount of collagen fibers (Fig. 4D). On the contrary, the subepicardial layer appears rich in collagen fibers and vessels (Fig. 4D). The bulbus is in continuity with the ventral aorta which presents a thick elastic wall (Fig. 4F), containing collagen fibers prevalently at the sub-endothelial and at the external layer of the tunica media (Fig. 4G). On the adventitia of the ventral aorta, a distinct vessel, belonging to the hypobranchial arterial system and surrounded by loose connective tissue, is evident (Fig. 4F,G). 3.2. eNOS immunodetection The immunolocalization of a phosphorylated eNOS-like isoform (for terminology and references in fish, see Imbrogno et al., 2011), obtained by using polyclonal anti p-eNOS (ser 1177) specific antibody, is shown in Fig. 5A,B. The enzyme is localized in the atrial (data not shown) and in the ventricular myocardium (Fig. 5A,B) of the goldfish heart. Labeling specificity was confirmed by the absence of the signal in parallel control sections treated without the primary antibody (Fig. 5C). In particular, p-eNOS was prevalently localized at the level of the vascular endothelium and, to a lesser extent, in the myocardiocytes and the endocardial endothelium (EE) (Fig. 5A,B).
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Fig. 4. Orcein (A) and Sirius red (B) staining of conus arteriosus. The thick fibrosa (ventricular layer of the conus valvar leaflets) rich in elastin (red arrows) and collagen (green arrows) is evident. (C) Detailed view of conus arteriosus; note the vessels (orange arrows) in the compact myocardium. Sirius red (D,G) and orcein (E,F) staining of bulbus longitudinal sections. In F and G, a transversal cut of the ventral aorta and the hypobranchial artery (black arrows) is evident. ca= conus arteriosus; sw = sinus wall; b = bulbus; v = ventricle; va= ventral aorta; Ep= epicardium; Sep = subepicardium; l = lumen.
3.3. Physiological results 3.3.1. Isolated working heart preparations By evaluating cardiac performance at different filling and afterload pressures, basal performance parameters were set up to a mean output pressure of approximately 1.5 kPa and cardiac output of about 10–12 mL/min/kg body mass. The basal hemodynamic parameters of isolated and perfused heart of C. auratus are reported in Table 1. Typical time-course curves of SV and HR indicated that the performance of the heart was stable for more than 2 h, after which the heart fell into a hypodynamic state characterized by a linear decrease in SV (data not shown). 3.3.2. Frank–Starling response The perfused C. auratus heart showed a typical Frank–Starling response. Increases of filling pressure (from 0.1 to 0.4 kPa) produced corresponding increases of SV. The maximum SV = 1.08 ± 0.09 mL/kg body mass was obtained with an input pressure of 0.4 kPa. Notably, we observed that the heart was not able to sustain afterload increases, as documented by the finding that values higher than 1.5 kPa resulted in a rapid decrease of cardiac performance (data not shown). 4. Discussion Although the evolution of the fish heart shows a high level of genetic conservation, the use of the same basic building blocks during its developmental processes has resulted in a remarkable variety of cardiac
designs (Olson, 2006). To some extent, such variety mirrors the impressive morpho-functional flexibility of the piscine cardiovascular system in relation to developmental and often extreme eco-physiological challenges associated with body growth and life style (Tota and Gattuso, 1996). Here we provide a morpho-functional characterization of the C. auratus four-chambered heart, i.e., sinus venosus, atrium, ventricle and bulbus, including the two distinct structures corresponding to the AV region and the conus arteriosus. The sinus venosus, which receives the peripheral venous blood to be ejected into the atrium, is made up of a thin wall and shows, in the proximity of the sino-atrial region, rings of nervous tissue, organized in cluster of ganglion cells probably corresponding to the primitive pacemaker region described in other teleosts (Yamauchi, 1980). In fishes, the atrium presents species-specific differences in both size and shape (Farrell and Jones, 1992). In C. auratus, the atrium appears as a large chamber completely trabeculated and rich in collagen and elastin. As extensively demonstrated by Icardo and Colvee (2011), the AV region, i.e. the area of the heart connecting the atrium and the ventricle, represents a distinct species-specific segment of the teleost heart, exhibiting relevant evolutionary diversification. The abundance of a vascularized compact myocardium in the C. auratus AV region represents a feature common to different teleosts with a Type II ventricle (Icardo and Colvee, 2011). In addition, as in other teleost species, also in C. auratus this region is surrounded by a ring of connective tissue formed by loose collagen fibers. According to the goldfish ventricle myoarchitecture and blood supply, the presence of both a vascularized compacta and a trabeculated
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Fig. 5. p-eNOS immunolocalization in the C. auratus ventricle (A,B). A strong signal appears on the vascular endothelium (yellow arrows). A weak signal is confined to the endocardial endothelium (red arrows) and in the myocardiocytes (pink arrows). Negative control is shown in C. In A and C nuclei are counterstained with Hoechst.
spongiosa allows to classify it as a Type II heart (Tota et al., 1983; Tota, 1989). The particular location of collagen fibers at the boundary between compacta and spongiosa suggests their possible role as a bonding glue to mechanically link the two differently oriented myocardial layers (Poupa et al., 1974; Tota, 1978; Icardo et al., 2005). In fact, while in the compacta the cardiomyocytes are oriented circumferentially, in the spongiosa they show a prevalent perpendicular trend. Conversely, in the salmonid heart, Pieperhoff et al. (2009) showed the absence of a continuous layer of collagen fibers and high amount of desmosomelike and fascia adherens-like structures that could function as anchorage structures between the compacta and the spongiosa. On the basis of these observations, it is conceivable that many structural features may concur to provide a morpho-functional boundary between the two muscular layers. Collagen fibers are also evident in the subepicardial layer where they can limit the extreme expansion of the ventricular chamber increasing its resilience (Icardo et al., 2005). Recent studies (Icardo et al., 2003; Icardo, 2006) demonstrated both in modern and in ancient teleosts the presence of a muscularized and vascularized conus arteriosus, interposed between the ventricle and the bulbus. We confirm its occurrence also in the goldfish heart where the conus, offering support to the valve complex, appears as a crown of tissue located at the base of the ventricle. The sinus valve appears as an element of continuity between ventricle, conus and bulbus. Moreover, for its abundance in elastin, which suggests a high deformability during cardiac cycle, it could play a role in facilitating heart mechanics. In fact, the high elasticity supports both the deformation during the dilation and the subsequent recovery of bulbus arteriosus; at the same time, by decreasing the conus stress, it favors valvar closure. In addition, the large amount of collagen and elastin of the thick ventricular fibrosa of the valve leaflets might exert a local resistance to the hemodynamic force applied on this side during the ventricular systole. The robust bulbus chamber contains a large amount of elastin, similar to that reported in carp (90%: Licht and Harris, 1973), which makes this cardiac chamber extremely flexible. In fish, the elevated compliance of the bulbus wall has been related to the high elastin/ collagen ratio (see for example the trout: Serafini-Fracassini et al., 1978). The gradual elastic recovery of the bulbus (windkessel effect: Farrell and Jones, 1992, and references therein), well evidenced by our functional experiments, ensures a constant blood flow towards the gills, preventing the damage of the delicate branchial vasculature as illustrated by Jones et al. (1993) in other teleosts. At the same time, the control of an excessive bulbus expansion during ventricular
systole may be guaranteed by high levels of collagen fibers, particularly localized in the subepicardium. The relationships between the structural design of the ventricular pump and its morphodynamic behavior have been largely investigated both in mammalian and non-mammalian vertebrates. In various fish species, this relationship, evaluated in terms of the relative contribution of pressure and volume work to the stroke work, allows a distinction between ventricles producing mainly volume work and those producing mainly pressure work (Tota and Gattuso, 1996; Icardo et al., 2005). C. auratus heart appears to exhibit a volume pump mechanical behavior. In fact, although under basal conditions (see Table 1), the CO resulted in 11.85 mL/min/kg, a value similar to those reported for other teleosts (Farrell and Jones, 1992), including the crucian carp (8.4 mL/min/kg at 8 °C; Farrell and Stecyk, 2007), the striking sensitivity to filling pressure changes, documented by the elevated SV values raised in response to increasing filling pressure, clearly indicates a hemodynamic ability to displace high blood volumes at low pressures. In this context, the elevated atrial/ventricular relative mass (27,6%), higher than that observed in other teleosts (e.g. eel: 18.4%, unpublished data), as well as the presence of large lacunary spaces, well comply with the mechanical behavior of the goldfish heart as volume pump. On the other hand, a typical volume pump heart is suited for maintaining constant the stroke volume only within a narrow range of afterloads. In fact, as highlighted by the Antarctic icefish paradigm (in vivo: Hemmingsen et al., 1972; in vitro: Tota et al., 1991), the output pressure values above which both cardiac work and power generation are quickly impaired result very low. Of note, in the in vivo crucian carp (C. carassius), which retains normal cardiac performance and autonomic cardiovascular regulation for at least 5 days of anoxia, the ventral aortic pressure is about 1.5 kPa (Stecyk et al., 2004). Interestingly, similarly to its relative crucian carp, the goldfish heart works at a very low output pressure and, in contrasts with several temperate teleosts able to sustain high values of afterloads [up to 5 kPa (Farrell and Jones, 1992; Tota and Gattuso, 1996)], showed an inability to maintain a constant systolic function in response to afterload increases. In turn, the latter caused an evident distension of the bulbus chamber, likely related to its high elastin/ collagen ratio. Whether the emptying constraint of the bulbus could be due to a particular fibrillar design of its wall remains to be clarified (for other teleost species see Braun et al., 2003). This behavior of C. auratus heart as volume pump is well evident when analyzed in the comparative context of the mechanical performance of
Table 1 Performance variables under basal conditions of isolated C. auratus heart preparations perfused with oxygen saturated Ringer's solution (n = 25). Heart rate beats min− 1
Filling pressure kPa
Output pressure kPa
Cardiac output ml min− 1 kg− 1
Stroke volume ml kg− 1
Power output mW g− 1
56.21 ± 3.24
0.11 ± 0.03
1.51 ± 0.05
11.85 ± 0.31
0.2 ± 0.02
0.36 ± 0.01
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Fig. 6. Values for afterload and stroke volume in elasmobranchs and teleosts under basal conditions. Bars represent the increases of afterload and stroke volume in conditions of maximum sustainable output or input pressures, respectively (modified from Tota and Gattuso, 1996).
different hearts, expressed in terms of pressure generation and volume movement, as illustrated in Fig. 6. In the fish heart, a widespread expression of a constitutive eNOSlike isoform, at vascular, endocardium endothelium and myocardiocytes levels, has been documented. Together with parallel physiopharmacological data, this points to a major role of the NOS/NO system in the fish heart (Imbrogno et al., 2011, and references therein). Consistent with our immunological findings, a key role of the NOS/NO system in the modulation of cardiac function in the goldfish heart may be hypothesized. Recent studies have highlighted the importance of the intracellular NO homeostasis during hypoxia in C. auratus heart (Hansen and Jensen, 2010; Pedersen et al., 2010; Jensen and Hansen, 2011). In particular, it has been supposed that when the NOS-mediated NO formation is compromised by a shortage of the substrate O2, an increase of tissue NOS expression to keep cellular NO levels stable may be expected (Hansen and Jensen, 2010). Studies are underway in our laboratory to clarify the putative cardiomodulatory role of the NOS/NO system as well as the existence of oxygen availability-related differences in the eNOS-like expression and localization in the goldfish heart. The present detection of intracardiac NOS in goldfish represents a prerequisite for this purpose. In conclusion, the striking goldfish tolerance against prolonged and severe hypoxia requires a balance between energy supply and demand, as well as coping with the acidosis associated with anaerobic end-product accumulation, i.e. lactic acidosis. This is prevented by goldfish through lactate conversion to ethanol and CO2, both excreted by the gills (Shoubridge and Hochachka, 1980; Bickler and Buck, 2007). Conceivably, from a morpho-functional standpoint, the volume pump ability of the goldfish heart to generate elevated CO and to displace in the branchial circulation a relatively large blood volume at low pressure may benefit the ethanol shuttling to the gills, thereby preventing its accumulation and even tissue intoxication. Moreover, the low values of PO, very similar to that estimated for the crucian carp in vivo (Farrell and Stecyk, 2007) and enough to be supported by anaerobic glycolysis, may ensure the goldfish heart to continue to beat during anoxic conditions. Acknowledgments This work was supported by “Ministero dell'Istruzione, dell'Università e della Ricerca” (e.g. MURST 60%) and by the Italian National Research Program in Antarctica (PNRA). The authors would like to thank Dr. E. Perrotta for electronmicroscopy technical assistance.
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