A novel technique for estimating the compact myocardium in fishes reveals surprising results for an athletic air-breathing fish, the Pacific tarpon

A method for quickly assessing the relative proportion of compact myocardium in the ventricle of teleosts is introduced and used in juvenile Pacific tarpon Megalops cyprinoides , a member of the only air-breathing elopomorph teleost genus. The proportion of compact myocardium increased with body mass, reaching up to 60% of the ventricular mass. The finding for tarpon was a surprising discovery since recent literature has suggested that air breathing evolved primarily as means of supplying oxygen to the fish heart during activity. The present data, which represent the first quantitative assessment of the compact myocardium for any air-breathing fish, suggest that myocardial oxygen supply in the tarpon is supplemented by the coronary circulation associated with compact myocardium during exercise, while air breathing is important during aquatic hypoxia. Compact myocardium was also measured as a point of reference in an extant representative from a more ancient fish lineage than the elopomorphs, the water-breathing spiny dogfish Squalus acanthias and found to be only 9% of ventricular mass. In conclusion, the presence of a coronary circulation in extant elasmobranchs may mean that the coronary circulation evolved well before air breathing in fishes and, for tarpon at least, the coronary oxygen supply to the ventricular myocardium has not necessarily been superseded by air breathing.     

Cardiac remodeling in fish: strategies to maintain heart function during temperature Change

Rainbow trout remain active in waters that seasonally change between 4°C and 20°C. To explore how these fish are able to maintain cardiac function over this temperature range we characterized changes in cardiac morphology, contractile function, and the expression of contractile proteins in trout following acclimation to 4°C (cold), 12°C (control), and 17°C (warm). The relative ventricular mass (RVM) of the cold acclimated male fish was significantly greater than that of males in the control group. In addition, the compact myocardium of the cold acclimated male hearts was thinner compared to controls while the amount of spongy myocardium was found to have increased. Cold acclimation also caused an increase in connective tissue content, as well as muscle bundle area in the spongy myocardium of the male fish. Conversely, warm acclimation of male fish caused an increase in the thickness of the compact myocardium and a decrease in the amount of spongy myocardium. There was also a decrease in connective tissue content in both myocardial layers. In contrast, there was no change in the RVM or connective tissue content in the hearts of female trout with warm or cold acclimation. Cold acclimation also caused a 50% increase in the maximal rate of cardiac AM Mg(2+)-ATPase but did not influence the Ca(2+) sensitivity of this enzyme. To identify a mechanism for this change we utilized two-dimensional difference gel electrophoresis to characterize changes in the cardiac contractile proteins. Cold acclimation caused subtle changes in the phosphorylation state of the slow skeletal isoform of troponin T found in the heart, as well as of myosin binding protein C. These results demonstrate that acclimation of trout to warm and cold temperatures has opposing effects on cardiac morphology and tissue composition and that this results in distinct warm and cold cardiac phenotypes.     

Preconditioning stimuli do not benefit the myocardium of hypoxia-tolerant rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss)</Emphasis>

In many vertebrates, a short episode of oxygen lack protects against myocardial necrosis during a subsequent, longer period of oxygen deprivation. This protective effect, termed preconditioning, also improves the functional recovery. Improved functional recovery has been reported for hypoxia-sensitive, in situ perfused rainbow trout hearts, but appears absent in another strain of rainbow trout that has a more hypoxia-tolerant heart. The results for the hypoxia-tolerant rainbow trout heart, however, might have occurred because the preconditioning stimuli were insufficient in either intensity or type to elicit cardioprotective effects. In the present study, we attempted to induce preconditioning in in situ perfused hearts from hypoxia-tolerant rainbow trout (Oncorhynchus mykiss), acclimated and tested at 10 °C, by either doubling the anoxic preconditioning stimulus (PO₂ of the perfusate <0.5 kPa) relative to earlier studies or by using short exposures to high concentrations of adrenaline. In addition, anoxic-preconditioning experiments were conducted at an acutely elevated temperature (15 °C) to increase myocardial sensitivity to oxygen lack. The effect of preconditioning stimuli was assessed by measuring cardiac performance before and after exposure to a 20-min anoxic challenge. In addition, myocardial condition was evaluated at the termination of the experiment by measuring myocardial concentrations of glycogen, high energy phosphates and lactate, as well as activities of pyruvate kinase and lactate dehydrogenase. Maximal cardiac performance in oxygenated control hearts was unchanged by the 2-h experimental protocol, whereas inclusion of a 20-min period of anoxia led to 25 and 35% reductions in maximal cardiac performance at 10 and 15 °C, respectively. Reduced contractility, however, could not be ascribed to myocardial necrosis, as the biochemical and energy state of the hearts was unaffected. Hence, anoxic exposure merely stunned the myocardium. At 10 °C, neither the anoxic nor adrenergic preconditioning protocols improved post-anoxic cardiac performance. Further, the preconditioning protocols did not reduce post-anoxic myocardial dysfunction at 15 °C, despite the increased cardiac sensitivity to anoxia at this temperature. Thus, despite using strong and different preconditioning stimuli compared with earlier studies, the cardio-protective effect of preconditioning seems to be absent in rainbow trout hearts that are inherently more hypoxia-tolerant.     

Coronary vascular volume remodelling in rainbow trout Oncorhynchus mykiss

The 34% increase in relative ventricular mass (M_rv) resulting from chronic anaemia (induced by an intraperitoneal injection of phenylhydrazine hydrochloride) was accompanied by a 117% increase in coronary vascular volume of diploid rainbow trout Oncorhynchus mykiss. Coronary vascular volume of normocythemic triploid fish was similar to that of normocythemic diploid fish despite a larger M_rv. These observations, in combination with previous studies, suggest that the vascularity of compact myocardium in O. mykiss can vary independently of M_rv.     

Heart structure of some deep-sea fish (Teleostei: Macrouridae)

The hearts of 29 species of macrourid teleosts were examined in this study. For the one species for which a length range was available ( Coryphaenoides (C.) rupestris ), the heart weight as a percentage of body weight was 0·059. This is similar to values for relatively inactive fish. The atrial myocardium was reduced and had only a sparse trabecular network. In some species it was surrounded by a highly developed epicardium, but in others there was interstitial connective tissue in the myocardium that may serve to strengthen this chamber. The ventricle was entirely spongy, and all species lacked an outer compact layer of myocardium and associated coronary vasculature. All the ventricles were sac-like in form. The bulbus arteriosus was highly complex, and in its proximal portion there was an endothelially-lined, inner tube surrounded by a spongy network of blood-filled spaces, outside which was an outer compact layer of smooth muscle and elastica. These features of the bulbus may prevent backflow of blood after ventricular systole. The endothelial cells lining the bulbus were usually PAS-positive and in some species contained acid mucopolysaccharides.     

Blood supply of the compact and spongy myocardium of fish, amphibia and reptiles]

Coronal arteries were injected with lead carbonate suspension and with Indian ink and cleared preparations 150--300 mkm thick were made in 195 hearts of fish, amphibians and reptiles and studied roentgenographically. It was stated that in Chondrichthyes (shark, skate) and in Chondrostei (beluga, stellate sturgeon, sturgeon), as well as in alligator both compact and spongy myocardium of the cardiac ventricle possess blood vessels. In teleostei, amphibians and reptiles (except alligator) spongy myocardium is avascular and receives its nutrition from the ventricle. In view of the data on the presence of blood vessels in the spongy myocardium in some vertebrates, it is impossible to accept the theory suggested by Grant and Regnier according to which vessels in the heart walls appear only in connection with compactization of the myocardium. Vascularization of the spongy myocardium is closely connected with oxygen saturation of the blood flowing through the heart. When this saturation is not satisfactory, the spongy myocardium has blood vessels. In alligator, vascularization of the spongy myocardium is connected with the fact that the heart has four chambers and there are arterial and venous blood streams.     

Cloning and characterization of glucose transporter in teleost fish rainbow trout (Oncorhynchus mykiss)

The facilitated diffusion of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUTs). In contrast to mammals, the glucose transport system of lower vertebrates remains unexplored. We detected glucose transport activity in rainbow trout embryos. Two GLUTs sharing 83% amino acid identity were cloned from juvenile fish, these have been denoted OnmyGLUT1A and OnmyGLUT1B. In adult trout OnmyGLUT1A is predominantly expressed in the heart with low expression in other tissues. An inverse terminal repeat of a Tc1-like transposable element was found in the 3'-untranslated region of OnmyGLUT1B. Phylogenetic analysis suggested that rainbow trout genes share a common ancestor with higher vertebrate GLUT1. We also found GLUT genes in several salmonid species.     

Ultrastructural study of the myocardium of the sinus venosus and sinoatrial valve in the dogfish (Scyliorhinus canicula)

The myocardium of the sinus venosus of the dogfish ( Scyliorhinus canicula ) is located between a thick subepicardial collagen-rich layer and a subendocardial network of nerve fibres and ganglion cells. The sinoatrial valve consists of two transversal folds of the cardiac wall which are separated by connective tissue, except in their free margins.
The myocardium of the sinus venosus and the sinusal face of the sinoatrial valve is arranged in bundles which are surrounded by a 40 nm-thick basal lamina. The myocardial cells measure about 7-9 μm in diameter at the nuclear level. Nerve terminals are frequent in the centre of the bundles. Most of the sinusal myocardiocytes have a scarce amount of myofibrils which are randomly orientated. The sarcoplasmic reticulum is relatively well developed and consists of peripheral couplings, subsarcolemmal vesicles, circular and longitudinal tubules. The scarce intercalated discs show only fasciae adhaerentes . Gap junctions, desmosomes or specific granules are not observed in the sinusal myocardiocytes of the dogfish. In contrast, the atrial myocardiocytes are smaller, about 5-6 μm in diameter at the nuclear level. The cytoplasm is denser and the myofibrils are abundant and orientated in parallel directions. Specific granules are present. although scarce. Subsarcolemmal vesicles are less frequent, while the atrial intercalated discs are larger and more abundant than those of the sinus venosus. Neural elements are scarce in the atrium.
The differences observed between sinus venosus and atrium might be related to the morphological criteria to distinguish between the nodal tissue and the working myocardiocytes of higher vertebrates. On the other hand, we think that the connective tissue placed between sinus venom and atrium means that the contraction impulse generated in the sinus venosus must reach the atrium through the free margin of the valve. It might play a role in the sinoatrial valve function.     

Ultrastructural morphometry of the myocardium of Thunnus alalunga

The common ventricle in the heart of the Thunnus alalunga was studied. The ventricular myocardium consists of an outer compact layer and a thick inner spongy layer. The compact layer has slightly larger cells (4-6 microns diameter) than the spongy layer (2.5-5 microns diameter). Ultrastructurally the myocardium displays normal arrangements of myofibrils and mitochondria. The sarcoplasmic reticulum is poorly developed. The intercalated discs are simple with the fascia adherens being the most frequent junctional type observed; occasionally a desmosome was seen. Nexus type junctions are present but are unassociated with the intercalated discs. There are no t-tubules evident but the plasmalemma exhibits numerous caveolae which rarely form couplings with the sarcoplasmic reticulum. A morphometric analysis of the volume percent of mitochondria and myofibrils showed that the myocardial cells in the spongy layer of the heart have a significantly greater volume percentage of mitochondria than the compact layer. No significant differences were found between myocardial regions when the volume percentages of myofibrils were compared. The physiological studies revealed that the albacore tuna has heart rates (120 bpm) and ventricular blood pressures (100 mmHg) that are among the highest reported for fish.     

Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.     

Homogenization modeling for the mechanics of perfused myocardium

Altered coronary perfusion can change the apparent diastolic stiffness of ventricular myocardium--the ‘garden hose’ effect. Our recent findings showed that myocardial strains are reduced during ventricular filling, primarily along the directions transverse to the coronary microvessels. In this article, we review hypotheses and theoretical models regarding the role that regional wall stress plays in the mechanical interaction between myocardium and coronary circulation. Various mechanisms have been used to explain the effects of the tissue stress on coronary flow, as well as the effect of coronary dynamics on myocardial mechanics. Many models of coronary pressure-flow relations using lumped parameter circuit analogs. Poroelasticity and swelling theories have been used to model the mechanics of perfused muscle. Here, we describe a new mathematical model of the mechanics of perfused myocardium derived using homogenization theory. In this model, perfused myocardium is treated as a nonlinear anisotropic elastic solid embedded with cylindrical vessels of known distensibility. The solid compartment is incompressible but the vascular compartment may change volume according to a simple relation between vessel diameter and perfusion pressure. The work done by the perfusion pressure in changing vascular volume contributes to the macroscopic strain energy and hence affects the stress and stiffness of the composite. Conversely, the stress in the tissue affects microvessel diameter and volume, since tractions transverse to the vessel axis oppose the internal blood pressure. Finite element simulations of passive filling show good agreement of model with experimental results.     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss)

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

Slackness between vessel and myocardium is necessary for coronary flow reserve

Tone regulation in coronary microvessels has largely been studied in isolated vessels in the absence of myocardial tethering. Here, the potential effect of radial tethering and interstitial space connective tissue (ISCT) between coronary microvessels and the surrounding myocardium was studied. We hypothesized that rigid tethering between microvessels and the myocardium would constrain the active contraction of arterioles and is not compatible with the observed tone regulation. The ISCT between coronary microvessels and myocardium in five swine was found to increase exponentially from 0.22 ± 0.02 μm in capillaries (modified Strahler order 0) of the endocardium to 34.9 ± 7.1 μm in epicardial vessels (order 10). Microvessels with both soft tethering and ISCT gap were capable of significant changes in vessel resistance (up to an ～1,600% increase), consistent with experimental measurements of high coronary flow reserve. Additionally, the mechanical energy required for myogenic contraction was estimated. The results indicate that rigid tethering requires up to four times more mechanical energy than soft tethering in the absence of a gap. Hence, the experimental measurements and model predictions suggest that effectiveness and efficiency in tone regulation can be achieved only if the vessel is both softly tethered to and separated from the myocardium in accordance with the experimental findings of ISCT gap. These results have fundamental implications on future simulations of coronary circulation.     

Immunohistochemical localization of bioactive peptides and amines associated with the chromaffin tissue of five species of fish

Biogenic peptides and amines associated with the chromaffin tissue in Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), spiny dogfish (Squalus acanthias) and Atlantic hagfish (Myxine glutinosa) were identified utilizing immunohistochemical techniques. Within the posterior cardinal vein (PCV) in cod, trout and eel, a subpopulation of chromaffin cells displayed immunoreactivity to tyrosine hydroxylase (TH) and dopamine--hydroxylase (DH) but not to phenylethanolamine-N-methyltransferase (PNMT). TH-like immunorectivity was observed within cells in hagfish hearts. Nerve fibres displaying vasoactive intestinal peptide (VIP) immunoreactivity and pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity innervated cod, trout and ell chromaffin cells. In eel, neuropeptide Y (NPY)-like and peptide YY (PYY)-like immunoreactivity was located within cells in the PCV, including chromaffin cells. Serotonin-like immunoreactivity was observed within eel and cod chromaffin cells and in hagfish hearts. In the dogfish axillary bodies, nerves displaying TH-like, VIP-like, PACAP-like, substance P-like and galanin-like immunoreactivity were observed. These results are compared with those of other vertebrates, and potential roles for these substances in the control of catecholamine release are suggested.     

Comparative and scaling aspects of heart and body weights with reference to blood supply of cardiac fibers

Relative heart weight (RHW) differs in vertebrates with the ratio 1:20 between extremes (bottom bound fishes--Pleuronectidae--and birds). When plotting heart weight (HW) against body weight (BW) one obtains channels which contain not only vertebrates of the same classes (poikilotherms, small and big mammals and birds) but also animals belonging to different classes: tuna fish data are located in the "small mammalian channel" together with data of large tropical snakes while large mammals (upwards 4000 g) belong to the "bird channel". Reasons for such groupings are not clear and physical activity seems not to be the only reason. When comparing active and non active vertebrates one finds that the RHW is as a rule greater in physically more active poikilotherms and homoiotherms. The RHW is also higher in wild than in domesticated forms the differences appearing after weaning (wild vs laboratory rat). In spongy type of myocardium the growth of cardiac fibers results in restriction of the blood flow through lacunae and the contact between endothelial cells lining growing strands of musculature probably provokes formation of capillaries. The appearance of mixed type of myocardium (outer compact and inner spongy compartments) is not bound to the water to land transition since it occurs also in some fishes; it does not occur or is rare in amphibia and is frequent in reptiles. The compact outer layer comprises a different proportion of the cardiac wall volume (5-73%). Metabolic differences were described between cardiac cells in compact and spongy compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of fish phagocytic cells by N-terminal peptides of proopiomelanocortin (NPP)

N-terminal peptide of proopiomelanocortin (NPP, or pro-gamma-MSH) has shown to exhibit biological activity such as stimulation of adrenal mitogenesis and prolactin release-inhibiting factor activity. Structurally, studies reveal a significant difference between fish NPP from that of tetrapods, as NPPs from carp and salmonid lack gamma-MSH. Thus, fish NPP may exhibit functions different from that of mammals. The activation of phagocytic cells by NPP was analysed using rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio. Rainbow trout and carp macrophages incubated with chum salmon NPP significantly enhanced the production of superoxide anion in comparison with control macrophages (without hormones). Both rainbow trout and carp macrophages had shown increased phagocytosis when stimulated administered with NPP. The above results were complemented by in vivo studies where NPP was administered to rainbow trout and carp. NPP significantly increased superoxide anion production as well as phagocytosis in macrophages. These results show that NPP in lower vertebrates activates the function of the phagocytic cells.