The Medical versus Zoological Concept of Outflow Tract Valves of the Vertebrate Heart.

The anatomical elements that in humans prevent blood backflow from the aorta and pulmonary artery to the left and right ventriclesare the aortic and pulmonary valves, respectively. Each valve regularly consists of three leaflets (cusps), each supported by its valvular sinus. From the medical viewpoint, each set of three leaflets and sinuses is regarded as a morpho-functional unit. This notion also applies to birds and non-human mammals. However, the structures that prevent the return of blood to the heart in other vertebrates are notably different. This has led to discrepancies between physicians and zoologists in defining what a cardiac outflow tract valve is. The aim here is to compare the gross anatomy of the outflow tract valvular system among several groups of vertebrates in order to understand the conceptual and nomenclature controversies in the field.

Experimental evidence of the genetic hypothesis on the etiology of bicuspid aortic valve aortopathy in the hamster model.

Bicuspid aortopathy occurs in approximately 50% of patients with bicuspid aortic valve (BAV), the most prevalent congenital cardiac malformation. Although different molecular players and etiological factors (genetic and hemodynamic) have been suggested to be involved in aortopathy predisposition and progression, clear etiophysiopathological mechanisms of disease are still missing. The isogenic (genetically uniform) hamster (T) strain shows 40% incidence of BAV, but aortic dilatations have not been detected in this model. We have performed comparative anatomical, histological and molecular analyses of the ascending aorta of animals with tricuspid aortic valve (TAV) and BAV from the T strain (TTAV and TBAV, respectively) and with TAV from a control strain (HTAV). Aortic diameter, smooth muscle apoptosis, elastic waviness, and Tgf-ß and Fbn-2 expression were significantly increased in T strain animals, regardless of the valve morphology. Strain and aortic valve morphology did not affect Mmp-9 expression, whereas Mmp-2 transcripts were reduced in BAV animals. eNOS protein amount decreased in both TBAV and TTAV compared to HTAV animals. Thus, histomorphological and molecular alterations of the ascending aorta appear in a genetically uniform spontaneous hamster model irrespective of the aortic valve morphology. This is a direct experimental evidence supporting the genetic association between BAV and aortic dilatation. This model may represent a population of patients with predisposition to BAV aortopathy, in which increased expression of Tgf-ß and Fbn-2 alters elastic lamellae structure and induces cell apoptosis mediated by eNOS. Patients either with TAV or BAV with the same genetic defect may show the same risk to develop bicuspid aortopathy.

Development of the ventricular myocardial trabeculae in Scyliorhinus canicula (Chondrichthyes): evolutionary implications.

The development of the ventricular myocardial trabeculae occurs in three steps: emergence, trabeculation and remodeling. The whole process has been described in vertebrates with two different myocardial structural types, spongy (zebrafish) and compact (chicken and mouse). In this context, two alternative mechanisms of myocardial trabeculae emergence have been identified: (1) in chicken and mouse, the endocardial cells invade the two-layered myocardium; (2) in zebrafish, cardiomyocytes from the monolayered myocardium invaginate towards the endocardium. Currently, the process has not been studied in detail in vertebrates having a mixed type of ventricular myocardium, with an inner trabecular and an outer compact layer, which is presumptively the most primitive morphology in gnathostomes. We studied the formation of the mixed ventricular myocardium in the lesser spotted dogfish (Scyliorhinus canicula, Elasmobranchii), using light, scanning and transmission electron microscopy. Our results show that early formation of the mixed ventricular myocardium, specifically the emergence and the trabeculation steps, is driven by an endocardial invasion of the myocardium. The mechanism of trabeculation of the mixed ventricular myocardium in chondrichthyans is the one that best reproduces how this developmental process has been established from the beginning of the gnathostome radiation. The process has been apparently preserved throughout the entire group of sarcopterygians, including birds and mammals. In contrast, teleosts, at least those possessing a mostly spongy ventricular myocardium, seem to have introduced notable changes in their myocardial trabeculae development.

Myosin heavy chain isoforms in the myocardium of the atrioventricular junction of Scyliorhinus canicula (Chondrichthyes, Carcharhiniformes).

The atrioventricular junction of the fish heart, namely the segment interposed between the single atrium and the single ventricle, has been studied anatomically and histologically in several chondrichthyan and teleost species. Nonetheless, knowledge about myosin heavy chain (MyHC) in the atrioventricular myocardium remains scarce. The present report is the first one to provide data on the MyHC isoform distribution in the myocardium of the atrioventricular junction in chondrichthyans, specifically in the lesser spotted dogfish, Scyliorhinus canicula, a shark species whose heart reflects the primitive cardiac anatomical design in gnathostomes. Hearts from five dogfish were examined using histochemical and immunohistochemical techniques. The anti-MyHC A4.1025 antibody was used to detect differences in the occurrence of MyHC isoforms in the dogfish, as the fast-twitch isoforms MYH2 and MYH6 have a higher affinity for this antibody than the slow-twitch isoforms MYH7 and MYH7B. The histochemical findings show that myocardium of the atrioventricular junction connects the trabeculated myocardium of the atrium with the trabeculated layer of the ventricular myocardium. The immunohistochemical results indicate that the distribution of MyHC isoforms in the atrioventricular junction is not homogeneous. The atrial portion of the atrioventricular myocardium shows a positive reactivity against the A4.1025 antibody similar to that of the atrial myocardium. In contrast, the ventricular portion of the atrioventricular junction is not labelled, as is the case with the ventricular myocardium. This dual condition suggests that the myocardium of the atrioventricular junction has two contraction patterns: the myocardium of the atrial portion contracts in line with the atrial myocardium, whereas that of the ventricular portion follows the contraction pattern of the ventricular myocardium. Thus, the transition of the contraction wave from the atrium to the ventricle may be established in the atrioventricular segment because of its heterogeneous MyHC isoform distribution. The findings support the hypothesis that a distinct MyHC isoform distribution in the atrioventricular myocardium enables a synchronous contraction of inflow and outflow cardiac segments in vertebrates lacking a specialized cardiac conduction system.

Embryonic development of bicuspid aortic valves.

Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, frequently associated with aortopathies and valvulopathies. The congenital origin of BAV is suspected to impact the development of the disease in the adult life. During the last decade, a number of studies dealing with the embryonic development of congenital heart disease have significantly improved our knowledge on BAV etiology. They describe the developmental defects, at the molecular, cellular and morphological levels, leading to congenital cardiac malformations, including BAV, in animal models. These models consist of a spontaneous hamster and several mouse models with different genetic manipulations in genes belonging to a variety of pathways. In this review paper, we aim to gather information on the developmental defects leading to BAV formation in these animal models, in order to tentatively explain the morphogenetic origin of the spectrum of valve morphologies that characterizes human BAV. BAV may be the only defect resulting from gene manipulation in mice, but usually it appears as the less severe defect of a spectrum of malformations, most frequently affecting the cardiac outflow tract. The genes whose alterations cause BAV belong to different genetic pathways, but many of them are direct or indirectly associated with the NOTCH pathway. These molecular alterations affect three basic cellular mechanisms during heart development, i.e., endocardial-to-mesenchymal transformation, cardiac neural crest (CNC) cell behavior and valve cushion mesenchymal cell differentiation. The defective cellular functions affect three possible morphogenetic mechanisms, i.e., outflow tract endocardial cushion formation, outflow tract septation and valve cushion excavation. While endocardial cushion abnormalities usually lead to latero-lateral BAVs and septation defects to antero-posterior BAVs, alterations in cushion excavation may give rise to both BAV types. The severity of the original defect most probably determines the specific aortic valve phenotype, which includes commissural fusions and raphes. Based on current knowledge on the developmental mechanisms of the cardiac outflow tract, we propose a unified hypothesis of BAV formation, based on the inductive role of CNC cells in the three mechanisms of BAV development. Alterations of CNC cell behavior in three possible alternative key valvulogenic processes may lead to the whole spectrum of BAV.

Bicuspid Aortic Valve in 2 Model Species and Review of the Literature.

Bicuspid aortic valve (BAV) is the most common human congenital cardiac malformation. Although the etiology is unknown for most patients, formation of the 2 main BAV anatomic types (A and B) has been shown to rely on distinct morphogenetic mechanisms. Animal models of BAV include 2 spontaneous hamster strains and 27 genetically modified mouse strains. To assess the value of these models for extrapolation to humans, we examined the aortic valve anatomy of 4340 hamsters and 1823 mice from 8 and 7 unmodified strains, respectively. In addition, we reviewed the literature describing BAV in nonhuman mammals. The incidences of BAV types A and B were 2.3% and 0.03% in control hamsters and 0% and 0.3% in control mice, respectively. Hamsters from the spontaneous model had BAV type A only, whereas mice from 2 of 27 genetically modified strains had BAV type A, 23 of 27 had BAV type B, and 2 of 27 had both BAV types. In both species, BAV incidence was dependent on genetic background. Unlike mice, hamsters had a wide spectrum of aortic valve morphologies. We showed interspecific differences in the occurrence of BAV between humans, hamsters, and mice that should be considered when studying aortic valve disease using animal models. Our results suggest that genetic modifiers play a significant role in both the morphology and incidence of BAV. We propose that mutations causing anomalies in specific cardiac morphogenetic processes or cell lineages may lead to BAV types A, B, or both, depending on additional genetic, environmental, and epigenetic factors.

Differential expression of myosin heavy chain isoforms in cardiac segments of gnathostome vertebrates and its evolutionary implications.

BACKGROUND: Immunohistochemical studies of hearts from the lesser spotted dogfish, Scyliorhinus canicula (Chondrichthyes) revealed that the pan-myosin heavy chain (pan-MyHC) antibody MF20 homogeneously labels all the myocardium, while the pan-MyHC antibody A4.1025 labels the myocardium of the inflow (sinus venosus and atrium) but not the outflow (ventricle and conus arteriosus) cardiac segments, as opposed to other vertebrates. We hypothesized that the conventional pattern of cardiac MyHC isoform distribution present in most vertebrates, i.e. MYH6 in the inflow and MYH7 in the outflow segments, has evolved from a primitive pattern that persists in Chondrichthyes. In order to test this hypothesis, we conducted protein detection techniques to identify the MyHC isoforms expressed in adult dogfish cardiac segments and to assess the pan-MyHC antibodies reactivity against the cardiac segments of representative species from different vertebrate groups. RESULTS: Western and slot blot results confirmed the specificity of MF20 and A4.1025 for MyHC in dogfish and their differential reactivity against distinct myocardial segments. HPLC-ESI-MS/MS and ESI-Quadrupole-Orbitrap revealed abundance of MYH6 and MYH2 in the inflow and of MYH7 and MYH7B in the outflow segments. Immunoprecipitation showed higher affinity of A4.1025 for MYH2 and MYH6 than for MYH7 and almost no affinity for MYH7B. Immunohistochemistry showed that A4.1025 signals are restricted to the inflow myocardial segments of elasmobranchs, homogeneous in all myocardial segments of teleosts and acipenseriforms, and low in the ventricle of polypteriforms. CONCLUSIONS: The cardiac inflow and outflow segments of the dogfish show predominance of fast- and slow-twitch MyHC isoforms respectively, what can be considered a synapomorphy of gnathostomes. The myocardium of the dogfish contains two isomyosins (MYH2 and MYH7B) not expressed in the adult heart of other vertebrates. We propose that these isomyosins lost their function in cardiac contraction during the evolution of gnathostomes, the later acquiring a regulatory role in myogenesis through its intronic miRNA. Loss of MYH2 and MYH7B expression in the heart possibly occurred before the origin of Osteichthyes, being the latter reacquired in polypteriforms. We raise the hypothesis that the slow tonic MYH7B facilitates the peristaltic contraction of the conus arteriosus of fish with a primitive cardiac anatomical design and of the vertebrate embryo.

The valves of the cardiac outflow tract of the starry ray, Raja asterias (Chondrichthyes; Rajiformes): Anatomical, histological and evolutionary aspects.

The cardiac outflow tract of chondrichthyans is composed of the myocardial conus arteriosus, equipped with valves at its luminal side, and the bulbus arteriosus devoid of myocardium. Knowledge of the histomorphology of the conal valves is scarce despite their importance in preventing blood backflow to the heart. Current information on the subject refers to a single shark species. The present report is the first to describe the structure of the conal valves of a batoid species, namely, Raja asterias. Hearts from seven starry rays were examined using scanning electron microscopy and histochemical techniques for light microscopy. In all hearts, the conus showed four transverse rows of three pocket-like valves each. Each valve was composed of a leaflet and its supporting sinus. The leaflet had a stout central body, rich in glycosaminoglycans, which contained fibroblasts, collagen and elastin. The central body was surrounded by two thin fibrous layers, outer and inner, formed mainly by collagen. The valves of the anterior row, which were the largest of the valvular system, were attached proximally to the conus arteriosus and distally to the bulbus arteriosus, and not to the ventral aorta as previously reported for chondrichthyans. The arrangement of the anterior valves in the starry ray is an anatomical pattern that apparently has been preserved throughout the evolution of vertebrates.

Cardiac, mandibular and thymic phenotypical association indicates that cranial neural crest underlies bicuspid aortic valve formation in hamsters.

Bicuspid aortic valve (BAV) is the most prevalent human congenital cardiac malformation. It may appear isolated, associated with other cardiovascular malformations, or forming part of syndromes. Cranial neural crest (NC) defects are supposed to be the cause of the spectrum of disorders associated with syndromic BAV. Experimental studies with an inbred hamster model of isolated BAV showed that alterations in the migration or differentiation of the cardiac NC cells in the embryonic cardiac outflow tract are most probably responsible for the development of this congenital valvular defect. We hypothesize that isolated BAV is not the result of local, but of early alterations in the behavior of the NC cells, thus also affecting other cranial NC-derived structures. Therefore, we tested whether morphological variation of the aortic valve is linked to phenotypic variation of the mandible and the thymus in the hamster model of isolated BAV, compared to a control strain. Our results show significant differences in the size and shape of the mandible as well as in the cellular composition of the thymus between the two strains, and in mandible shape regarding the morphology of the aortic valve. Given that both the mandible and the thymus are cranial NC derivatives, and that the cardiac NC belongs to the cephalic domain, we propose that the causal defect leading to isolated BAV during embryonic development is not restricted to local alterations of the cardiac NC cells in the cardiac outflow tract, but it is of pleiotropic or polytopic nature. Our results suggest that isolated BAV may be the forme fruste of a polytopic syndrome involving the cranial NC in the hamster model and in a proportion of affected patients.

Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1ß, G3pdh and Polr2a. We propose the use of Cdkn1ß, or both Cdkn1ß and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

Unusual anatomical origins of the coronary arteries in C57BL/6 mice. Are they strain-specific?

A previous manuscript [Fernández B, et al. (2008) J Anat 212, 12] reported on the unusual coronary artery patterns in mice belonging to the C57BL/6 strain. The aim here was to elucidate whether this pattern is unique to C57BL/6 mice or appears in other laboratory mouse strains and in wild-living mice. Stereomicroscopy, scanning electron microscopy, light microscopy and a corrosion cast technique were used to examine 597 adult mice belonging to three inbred strains (C57BL/6, Balb/c, DBA/2), three outbred stocks (CD1, OF1, NMRI) two hybrid lines (129sv × BL/6, CD2F1) and wild mice. It was shown that lock-like ostium is an exclusive trait of C57BL/6 mice, whereas left septal artery, accessory ostium, high take-off, intramural course and solitary ostium in aorta are all present in the different laboratory strains and wild mice included in the present study. However, each mouse population shows a specific incidence of these coronary conditions. Several clinically relevant human coronary artery anomalies are present in healthy mice from different strains that may serve as animal models for humans. These results should be taken into consideration in research concerning the murine coronary system, especially in coronary artery occlusion experiments and in studies on cardiovascular developmental biology using murine mutant lines.

Quadricuspid aortic valves in Syrian hamsters and their formation according to current knowledge on valvulogenesis.

Occurrence of quadricuspid aortic valves has been reported in humans, in nine dogs and in a greater white-toothed shrew. Moreover, two cases of developing aortic valves with four anticipated leaflets have been described in Syrian hamster embryos. Currently, however, no case of quadricuspid aortic valve in adult hamsters has been recorded. The aim here is to present four adults of this rodent species, two of them with unequivocally quadricuspid aortic valves and the other two with quadricuspid-like aortic valves. The four anomalous aortic valves were detected among 4,190 Syrian hamsters examined in our laboratory, representing an incidence of 0.09%. None of the affected hamsters showed apparent signs of disease. The present findings are considered on the light of current empirical knowledge about the morphogenesis of quadricuspid and bicuspid aortic and pulmonary valves. Quadricuspid aortic valves result from the partition of one of the normal mesenchymal cushions which normally give rise to normal (tricuspid) valves, while quadricuspid-like valves might be the product of a combined mechanism of fusion and partition of the cushions at the onset of the valvulogenesis. The presence of aortic valves with four leaflets in ancient mammalian lineages such as insectivors and rodents suggest that quadricuspid aortic valves, although showing almost certainly a low incidence, may be widespread among the different groups of mammals, including domestic animals.

Osteoglycin deficiency does not affect atherosclerosis in mice.

OBJECTIVE: The small leucine-rich proteoglycan Osteoglycin/Mimecan (OGN) is a component of the extracellular matrix, where it regulates collagen fibrillogenesis and cytokine availability. OGN is abundant in normal vessels and in atherosclerotic and restenotic lesions of rat, rabbit and human arteries. Osteoglycin-null mice show alterations in the thickness of collagen fibers of the cornea and the skin. In this work, we inspect the possible involvement of OGN in the atherosclerosis progression using a double knockout mouse model. METHODS: In order to examine the progression of atherosclerosis in the absence of OGN, we developed double Apoe and Ogn knockout mice and performed a comparative histomorphological and immunofluorescence study of the atherosclerotic lesions of Apoe(-/-)Ogn(-/-) and Apoe(-/-)Ogn(+/+) mice. RESULTS: We demonstrate the presence of Ogn transcript in the aorta of wildtype mice, its absence in Ogn(-/-) mice, and the normal histomorphology of arteries of Ogn(-/-) mice. The composition of the extracellular matrix and also the cellular content and distribution were similar in atherosclerotic lesions of Apoe(-/-)Ogn(-/-) and Apoe(-/-)Ogn(+/+) mice. Quantification of the lesion size revealed no significant differences between double and single knockout mice. The incidence, size and distribution of calcium deposits were similar in both groups of mice. CONCLUSIONS: The lack of the proteoglycan OGN does not affect the progression of atherosclerosis in mice. Possible causes for the absence of phenotype in the Apoe/Ogn double mutants are discussed.

Chronotopographical distribution patterns of cell death and of lectin-positive macrophages/microglial cells during the visual system ontogeny of the small-spotted catshark Scyliorhinus canicula.

The patterns of distribution of TUNEL-positive bodies and of lectin-positive phagocytes were investigated in the developing visual system of the small-spotted catshark Scyliorhinus canicula, from the optic vesicle stage to adulthood. During early stages of development, TUNEL-staining was mainly found in the protruding dorsal part of the optic cup and in the presumptive optic chiasm. Furthermore, TUNEL-positive bodies were also detected during detachment of the embryonic lens. Coinciding with the developmental period during which ganglion cells began to differentiate, an area of programmed cell death occurred in the distal optic stalk and in the retinal pigment epithelium that surrounds the optic nerve head. The topographical distribution of TUNEL-positive bodies in the differentiating retina recapitulated the sequence of maturation of the various layers and cell types following a vitreal-to-scleral gradient. Lectin-positive cells apparently entered the retina by the optic nerve head when the retinal layering was almost complete. As development proceeded, these labelled cells migrated parallel to the axon fascicles of the optic fiber layer and then reached more external layers by radial migration. In the mature retina, lectin-positive cells were confined to the optic fiber layer, ganglion cell layer and inner plexiform layer. No evident correlation was found between the chronotopographical pattern of distribution of TUNEL-positive bodies and the pattern of distribution of lectin-labelled macrophages/microglial cells during the shark's visual system ontogeny.

Retinal histogenesis and cell differentiation in an elasmobranch species, the small-spotted catshark Scyliorhinus canicula.

Here we present a detailed study of the major events in the retinal histogenesis in a slow-developing elasmobranch species, the small-spotted catshark, during embryonic, postnatal and adult stages using classical histological and immunohistological methods, providing a complete neurochemical characterization of retinal cells. We found that the retina of the small-spotted catshark was fully differentiated prior to birth. The major developmental events in retinal cell differentiation occurred during the second third of the embryonic period. Maturational features described in the present study were first detected in the central retina and, as development progressed, they spread to the rest of the retina following a central-to-peripheral gradient. While the formation of both plexiform layers occurs simultaneously in the retina of the most common fish models, in the small-spotted catshark retina the emergence of the outer plexiform layer was delayed with respect to the inner plexiform layer. According to the expression of the markers used, retinal cell differentiation followed a vitreal-to-scleral gradient, with the exception of Müller cells that were the last cell type generated during retinogenesis. This vitreal-to-scleral progression of neural differentiation seems to be specific to slow-developing fish species.

Phylogeny informs ontogeny: a proposed common theme in the arterial pole of the vertebrate heart.

In chick and mouse embryogenesis, a population of cells described as the secondary heart field (SHF) adds both myocardium and smooth muscle to the developing cardiac outflow tract (OFT). Following this addition, at approximately HH stage 22 in chick embryos, for example, the SHF can be identified architecturally by an overlapping seam at the arterial pole, where beating myocardium forms a junction with the smooth muscle of the arterial system. Previously, using either immunohistochemistry or nitric oxide indicators such as diaminofluorescein 2-diacetate, we have shown that a similar overlapping architecture also exists in the arterial pole of zebrafish and some shark species. However, although recent work suggests that development of the zebrafish OFT may also proceed by addition of a SHF-like population of cells, the presence of a true SHF in zebrafish and in many other developmental biological models remains an open question. We performed a comprehensive morphological study of the OFT of a wide range of vertebrates. Our data suggest that all vertebrates possess three fundamental OFT components: a proximal myocardial component, a distal smooth muscle component, and a middle component that contains overlapping myocardium and smooth muscle surrounding and supporting the outflow valves. Because the middle OFT component of avians and mammals is derived from the SHF, our observations suggest that a SHF may be an evolutionarily conserved theme in vertebrate embryogenesis.