Incidence and type of bicuspid aortic valve in two model species

Incidence and type of bicuspid aortic valve in two model species. MC Fernández 1,2, A López-García 1,2, MT Soto 1, AC Durán 1,2 and B Fernández 1,2. 1 Department of Animal Biology, Faculty of Science, University of Málaga, Spain. 2 Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain. Bicuspid aortic valve (BAV) is the most frequent human congenital cardiac malformation, with an incidence of 1–2% worldwide. Two morphological types exist: type A (incidence 0.75–1.25%) and type B (incidence 0.25–0.5%), each with a distinct aetiology and natural history. Currently, ten animal models of BAV have been described in two different rodent species: one spontaneous Syrian hamster (Mesocricetus auratus) model of BAV type A and nine mutant laboratory mouse (Mus musculus) models of BAV type B. It remains to be elucidated whether the mutations leading to BAV in these models are typespecific or whether there are inter-specific differences regarding the type of BAV that hamsters, mice and humans may develop. To solve this issue, we have characterized the incidence and types of BAVs in four inbred, two outbred and two hybrid lines of Syrian hamsters (n=4,340) and in three inbred, three outbred and one hybrid lines of laboratory mice (n=1,661) by means of stereomicroscopy and scanning electron microscopy. In addition, we have reviewed and calculated the incidence and type of BAVs in the published papers dealing with this anomaly in mice. Our results indicate that the Syrian hamster develops BAVs type A and B including a variety of morphologies comparable to those of humans, whereas the mouse develops only BAVs type B with a short spectrum of valve morphologies. Thus, inter-specific differences between human and mouse aortic valves must be taken into consideration when studying valve disease in murine models. This work was supported by P10-CTS-6068.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. P10-CTS-6068

Contribution of Xenopus model to a better understanding of cardiac outflow tract

Contribution of Xenopus model to a better understanding of cardiac outflow tract. A Torres-Prioris 1, SJ Smith 2, TJ Mohun 2, B Fernández 1, AC Durán 1. 1 Department of Animal Biology, Faculty of Science, and Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain. 2 Developmental Biology Division, The Francis Crick Institute, Mill Hill Laboratory, London, UK. The morphology and morphogenesis of the cardiac outflow tract is a major topic in the study of the vertebrate circulatory system, especially regarding the pathologies affecting this region in humans. Recent studies have demonstrated that, in fish, the cardiac outflow tract consists of a myocardial conus arteriosus and a nonmyocardial bulbus arteriosus. Moreover, the bulbus arteriosus of fish has been considered homologous to the intrapericardial base of the aortic and pulmonary trunks of birds and mammals. Under this perspective, we have conducted a study on the outflow tract of Xenopus laevis, using histological, immunohistochemical and 3D reconstruction techniques. It has been assumed that the outflow tract of Xenopus, which is intercalated between the ventricle and the great arterial trunks, is of myocardial nature. At its luminal side, it contains two sets of valves between which the so-called spiral valve lies. Our results demonstrate that, together with a proximal myocardial segment, a distal, nonmyocardial, intrapericardial segment is also present in amphibians. We propose that this distal segment, from which the pulmocutaneous and systemic arteries arise, is homologous to the bulbus arteriosus of fish. Therefore, the bulbus arteriosus is an evolutionarily conserved structure, which has become the aortic and pulmonary roots of birds and mammals. Our findings contribute to strengthening Xenopus as a good model to better understand the outflow tract morphology and evolution, and as an emerging model for studying human congenital heart diseases. This work was supported by CGL2010-16417, BES-2011-046901, Estancias Breves para FPI (2012, 2013) and FEDER funds.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. CGL2010-16417, BES-2011-046901, Estancias Breves para FPI (2012, 2013), FEDER funds

Is the bulbus arteriosus of fish homologous to the mamalian intrapericardial thoracic arteries?

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013.Anatomical Record, Volume 296, Special Feature — 1: P-089.Two major findings have significantly improved our understanding of the embryology and evolution of the arterial pole of the vertebrate heart (APVH): 1) a new embryonic presumptive cardiac tissue, named second heart field (SHF), forms the myocardium of the outflow tract, and the walls of the ascending aorta (AA) and the pulmonary trunk (PT) in mammals and birds; 2) the bulbus arteriosus (BA), previously thought to be an actinopterygian apomorphy, is present in all basal Vertebrates, and probably derives from the SHF. We hypothesized that the intrapericardial portions of the AA and the PT of mammals are homologous to the BA of basal vertebrates. To test this, we performed 1) a literature review of the anatomy and embryology of the APVH; 2) novel anatomical, histomorphological, and embryological analyses of the APVH, comparing basal (Galeus atlanticus), with apical (Mus musculus and Mesocricetus auratus) vertrebrates. Evidence obtained: 1) Anatomically, BA, AA, and PT are muscular tubes into the pericardial cavity, which connect the distal myocardial outflow tracts with the aortic arch system. Coronary arteries run through or originate at these anatomical structures; 2) Histologically, BA, AA, and PT show an inner layer of endothelium covered by circumferentially oriented smooth muscle cells, collagen fibers, and lamellar elastin. The histomorphological differences between the BA and the ventral aorta parallel those between intrapericardial and extrapericardial great arteries; 3) Embryologically, BA, AA, and PT are composed of smooth muscle cells derived from the SHF. They show a similar mechanism of development: incorporation of SHF‐derived cells into the pericardial cavity, and distal‐to‐proximal differentiation into an elastogenic cell linage. In conclusion, anatomical, histological and embryological evidence supports the hypothesis that SHF is a developmental unit responsible for the formation of the APVH. The BA and the intrapericardial portions of the great arteries must be considered homologous structures.Proyecto P10-CTS-6068 (Junta de Andalucía); proyecto CGL-16417 (Ministerio de Ciencia e Innovación); Fondos FEDER

Different laboratory mouse strains show distinct coronary artery patterns

Different laboratory mouse strains show distinct coronary artery patterns. MC Fernández 1,2, A López-García 1,2, M Lorenzale 1, V Sans-Coma 1,2, AC Durán 1,2 and B Fernández 1,2. 1 Department of Animal Biology, Faculty of Science, University of Málaga, Spain. 2 Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain. The C57BL/6 (BL/6) mouse strain is one of the most common models in research involving laboratory animals, particularly on studies of the cardiovascular system. It has been reported (Fernandez B, et al. J Anat 2008 212(1):12–18) that this strain presents an unusual coronary artery (CA) pattern, including congenital CA anomalies, which are clinically relevant in humans. The aim of the present study was to elucidate whether this pattern is strain-specific or appears in other mouse populations. We used stereomicroscopy, scanning electron microscopy, light microscopy, and a corrosion cast technique in 597 adult mice belonging to three inbred strains (BL/6, Balb/c, DBA/2), three outbred stocks (CD1, OF1, NMR1), two hybrid lines (129sv x BL/6, CD2F1) and wild mice. Lock-like ostium was only detected in BL/6 mice, whereas left septal artery, accessory ostium, high take-off, intramural course, and solitary ostium of one CA in aorta were present in different laboratory strains and in wild mice. However, each mouse population showed a specific incidence of these coronary conditions. These results should be taken into account when studying the murine coronary system, especially in CA occlusion experiments and in studies on cardiovascular development involving murine mutant lines. In addition, we propose that several laboratory mouse strains may serve as appropriate animal models to study several clinically relevant human congenital anomalies of the CAs. Our results suggest that some of these CA anomalies are subject to a simple mode of inheritance. This work was supported by P10-CTS-6068 and PI- 0888-2012.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. P10-CTS-6068. PI-0888-201

DISTRIBUTION OF PIGMENT CELLS IN THE HEART OF THE RABBITFISH, CHIMAERA MONSTROSA (CONDRICHTHYES: HOLOCEPHALI)

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013. Anatomical Record, Volume 296, Special Feature — 1: P-076.The study of extracutaneous cells producing and storing melanin is of interest because it may provide valuable information about the presence of neural crest elements in internal organs and tissues. Here we report, for the first time, the presence and distribution of melanophores in the heart of a chondrichthyan species, the rabbitfish, Chimaera monstrosa. Pigment cells were found in all of 20 hearts examined. Pigment cells occur mainly in the cardiac outflow tract, which consists of two anatomical components, the proximal, myocardial conus arteriosus and the distal, non-myocardial bulbus arteriosus. A few groups of dark pigmented cells were found in the apex of the ventricle of one specimen and in the atrium of two specimens. In all instances, the melanophores were located in the subepicardial space, where they could be well recognized in both unstained and stained histological sections. The distribution and intensity of the pigmentation in the cardiac outflow tract varies markedly between individuals. In all cases, however, the pigmented area is larger on the dorsal than on the ventral surface. Dorsally, the size of the pigmented area ranges from a fringe that includes the bulbus and the distal part of the conus to the whole surface of the outflow tract. Ventrally, the pigmented area does not cover the entire conus arteriosus. The intensity of the pigmentation also varies widely; in general, it is highest at the distal portion of the conus. There is no relationship between the distribution and intensity of the pigmentation and the sex and age of the animals. The functional role of the pigmented cells is unknown. If the melanophores in the heart of C. monstrosa are indeed of neural crest origin, it would suggest a notable contribution of the neural crest cells to the cardiac outflow tract in holocephalans.Proyecto CGL2010-16417/BOS; Fondos FEDER BES-2011-04690

PIGMENTATION OF THE HEART IN THE BICHIR, POLYPTERUS SENEGALUS

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013. Anatomical Record, Volume 296, Special Feature — 1: P-078.The presence of melanin-containing cells in the heart has been documented in tetrapods, but not in fish. It has been even suggested that dark pigmented cells are exclusively associated with hearts having two atria and two ventricles. The aim here is to report the occurrence of pigment cells in the heart of the bichir, an extant representative of the polypteriformes, an ancient ray-finned fish lineage that split from the stem of the actinopterygians soon after their divergence from the sarcopterygians. The bichir heart is composed of sinus venosus, atrium, ventricle, conus arteriosus and bulbus arteriosus arranged sequentially within the pericardial cavity. Dendritic-shaped cells containing melanosomes were found in the five cardiac components of the 12 bichirs included in this study. Numerous melanophores were distributed regularly over the surface of all segments having myocardium in their walls, thus resulting in a marked pigmentation of the whole heart. The bulbus arteriosus, which in the bichir is reduced in size, showed an even more intense pigmentation. In all instances, the melanophores were localized in the subepicardial space. Pigment cells also occurred in the pericardium and ventral aorta. The functional role of melanocytes in the tetrapod heart remains obscure. Antiinflamatory activity, cytoprotection and effects on the viscoelastic properties of the cardiac tissue have been adduced as possible actions of such cells. The role of pigment cells in the bichir heart constitutes a new open question. Interestingly, however, the only cells that have been shown to form melanin-containing cells in the heart derive from the neural crest. If the melanophores of the bichir heart are indeed of neural crest origin, it would suggest a much more extensive contribution and persistence of elements from the neural crest in the primitive heart of jawed vertebrates as assumed so far in most papers devoted to vertebrate heart embryology.Proyecto CGL2010-16417/BOS; Fondos FEDER; Beca FPI ref. BES-2011-046901

The bulbus arteriosus of the holocephalan heart

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013.Anatomical Record, Volume 296, Special Feature — 1: P-074.Previous work has shown that the outflow tract of the elasmobranch heart, namely the cardiac portion intercalated between the ventricle and the ventral aorta, does not consist of a single component, the conus arteriosus, as has classically been assumed, but two, the myocardial conus arteriosus and the non-myocardial bulbus arteriosus. From the evolutionary perspective, knowledge of the anatomy of the cardiac outflow tract of the holocephali is important, as they are the sister group of elasmobranchs. Our aim is to describe the cardiac outflow tract of four holocephalan species, two of them, Chimaera monstrosa and Hydrolagus affinis of the family Chimaeridae, and the other two, Harriotta raleighana and Rhinochimaera atlantica, of the family Rhinochimaeridae. The cardiac outflow tract of the four species consisted of a myocardial conus arteriosus, furnished with valves, and a bulbus arteriosus devoid of cardiac muscle. Both the bulbus and conus are tubular in shape. The length of the bulbus relative to the total length of the outflow tract is somewhat smaller in the rhinochimaerids (15%-19%) than in the chimaerids (19%-23%). The bulbus is covered by epicardium and is crossed by the main coronary artery trunks. Histologically, the bulbus is mainly composed of elastin and collagen, and, to a lesser extent, by smooth muscle. This suggests that in holocephalans, the bulbus actively helps to protect the gill vasculature from exposure to high-pressure pulses of blood. Our results prove that the bulbus arteriosus is common to chondrichthyans. In addition, they support the hypothesis that the cardiac outflow tract consisted of a conus arteriosus and a bulbus arteriosus from the beginning of the jawed vertebrate radiation, contributing to our understanding of the morphological changes that have occurred at the arterial pole of the heart in both actinopterygians and sarcopterygians.Proyecto CGL2010-16417/BOS; Fondos FEDE

MYOCARDIAL STRUCTURE AND VASCULARIZATION OF THE HEART VENTRICLE IN HOLOCEPHALI: IMPLICATIONS FOR HEART EVOLUTION

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013. Anatomical Record, Volume 296, Special Feature — 1: P-075.It has been classically assumed that the ventricle of the primitive vertebrate heart is composed of spongy myocardium, supplied exclusively by oxygen-poor, luminal blood. This idea is on two facts: (1) extant agnathans have a spongy ventricular myocardium, and (2) in avian and mammalian embryos, the formation of trabeculated myocardium precedes the appearance of compact myocardium. Recently, it has been proposed that, like elasmobranchs, the early gnathostomes possess a fully vascularised ventricle composed of mixed myocardium. We tested this idea by studying the structure and vascularisation of the ventricular myocardium in four holocephalan species of the families Chimaeridae and Rhinochimaeridae. Chimaera monstrosa, Hidrolagus affinis and Harriotta raleighana have a spongy myocardium covered by a thin layer of cardiac muscle. In H. raleighana, the compacta is reduced to an extremely fine rim. In all three species there is a well-developed coronary artery system consisting of subepicardial vessels which give off branches that penetrate the myocardial trabeculae. Rhinochimaera atlantica has no compacta and its ventricular coronary artery system is reduced to subepicardial vessels that do not enter the spongy layer. This report is the first to show that in wild living vertebrates, a coronary artery system supplying the whole myocardium exists in the absence of a well-developed compacta, which supports experimental work that shows that myocardial cell proliferation and coronary vascular growth rely on genetically separated programs. We conclude that the mixed ventricular myocardium is primitive for chondrichthyans, and that the lack of compacta in some holocephalans is a derived character. Moreover our results support the hypotheses that the mixed myocardium is the primitive condition in gnathostomes, and that the absence of a compacta in different actinopterygian taxa is the result of its repeated loss during evolution.Proyecto CGL2010-16417/BOS; Fondos FEDE

The Myosin Heavy Chain specific A4.1025 antibody discriminates different cardiac segments in ancient groups of gnathostomes: Morphological and evolutionary implications

El resumen aparece en el Program & Abstracts of the 11th International Congress of Vertebrate Morphology, Washington DC 2016. Anatomical Record, Volume 299, Special Feature: 263.The pan-Myosin Heavy Chain (pan-MyHC) marker MF20 have been reported to show similar, homogeneous signal in the myocardial segments of the heart of teleosts and tetrapods. However, in an ongoing study of the myocardial structure of the dogfish (Scyliorhinus canicula; Chondrichthyes), we observed differential immunostaining of the cardiac segments using another pan-MyHC, the A4.1025 antibody. In order to investigate the relevance of this finding for better understanding of the morphology and evolution of the vertebrate heart, we performed immunohistochemistry, slot blot and western blot in several species of chondrichthyans, actinopterygians and mammals using the above mentioned antibodies. In the dogfish heart, A4.1025 and MF20 specifically recognized MyHC isoforms, although with different degree of affinity. MF20 reactivity was homogeneous and high in all the myocardial segments. However, A4.1025 reactivity was heterogeneous. It was high in the sinus venosus (external layer), atrium and atrioventricular region, low in the ventricle and conus arteriosus, and null in the internal layer of the sinus venosus. A heterogeneous pattern of A4.1025 immunoreactivity was also detected in two other elasmobranchs, a holocephalan, a polypteryform and an acipenseriform. In all of these species, MF20 immunoreactivity was homogeneous. In addition, both markers showed a homogeneous immunoreactivity pattern in teleosts and mammals. Our results indicate that in the hearts of ancient gnathostomes, in all of which a conspicuous conus arteriosus exists, one or more MyHC isoforms with low affinity for A4.1025 show segment-specific distributions. Thus, A4.1025 appears to be an appropriated marker to identify the cardiac segments and their boundaries. We propose that the segmentspecific distribution of MyHC isoforms may generate a particular type of myocardial contractility associated with the presence of a conus arteriosus.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. CGL2014-52356-P, CEIMAR, BIO 203, FEDE

Effect of hyperlipidic diets on normal and abnormal aortic valves in the Syrian hamster: A preliminary study

Effect of hyperlipidic diets on normal and abnormal aortic valves in the Syrian hamster: A preliminary study. MC Fernández 1,2, J Moncayo-Arlandi 1, MT Soto 1, MA López-Unzu 1, B Fernández 1,2 and AC Durán 1,2. 1 Department of Animal Biology, Faculty of Science, University of Málaga, Spain. 2 Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain. Bicuspid aortic valve (BAV) is the most frequent human congenital cardiac malformation. It frequently becomes stenotic due to calcification by an atherosclerosis-like process. Hyperlipidic diets have been classically used to induce atherosclerosis in laboratory animals, including Syrian hamsters. The aim here is to evaluate the effect of hyperlipidic diets in hamsters having different incidence of BAVs. We used a unique inbred strain of Syrian hamsters with a high ( 40%) incidence of spontaneous BAV, morphologically similar to that in man, another inbred strain with a low ( 4%) incidence of BAV, and an outbred, second control line, acquired from Charles River Laboratories. Three experimental groups were fed with standard diet supplemented with 2% cholesterol plus 15% butter during five months. In parallel, three control groups were fed with unmodified standard diet. Hyperlipidic diets induced lesions in the aortic valve and ascending aortic wall, i.e. subendothelial lipid deposits, valve sclerosis, and neo-intima in the aorta. We performed a preliminary, qualitative, comparative study of the lesions associated with the different animal populations and valvular phenotypes. Our results indicate that (1) the type and severity of the lesions varied among the three hamster populations, suggesting that genetic factors may be involved; (2) the aortic valve morphology seems not to determine the severity of the valvular lesions. We conclude that our hamster strain with high incidence of BAV is a promising animal model for studies on human aortic stenosis. This work was supported by P10-CTS-6068.Universidad de Málaga. Campus de Excelencia Andalucía Tech. P10-CTS-6068