Signal molecule changes in the gills and lungs of the African lungfish Protopterus annectens, during the maintenance and arousal phases of aestivation.

African lungfishes are obligate air breathers, with reduced gills and pulmonary breathing throughout their life. During the dry season they aestivate on land, with the collapse of secondary lamellae of their gills and the establishment of an exclusive aerial ventilation through the vascularization and expansion of their lungs. To date, the mechanisms underlining the respiratory organ remodeling in aestivating lungfishes are unknown. This study aimed to identify key switch components of the stress-induced signal transduction networks implicated in both rapid and medium-long term remodeling of the gills and lungs of the African lungfish Protopterus annectens during aestivation. Through immunofluorescence microscopy and Western blotting, the localization and the expression of nitric oxide synthase (NOS), Akt, Hsp-90 and HIF-1α were evaluated in both gills and lungs exposed to three experimental conditions: freshwater (FW), 6 months of experimentally induced aestivation (6mAe), and 6 days after arousal from 6 months of aestivation (6mAe6d). After 6mAe, the expression of NOS (p-eNOS antibody), Akt (p-Akt antibody), and Hsp-90 decreased in the gills, while NOS and Hsp-90 expression increased with Akt remained unchanged in the lungs. Upon 6mAe6d, NOS, Akt and Hsp-90 expression in the gills returned to the respective FW values. In the lungs of the aroused fish, NOS and Akt decreased to their respective FW levels, while Hsp-90 expression was enhanced with respect to aestivation. In both respiratory organs, the qualitative and quantitative patterns of HIF-1α expression correlated inversely to those of NOS. Overall, our findings suggest that the molecular components of the NOS/NO system changed in a tissue-specific manner in parallel with organ readjustment in the gills and lungs of P. annectens during aestivation and arousal.

Determination of the mechanical properties of normal and calcified human mitral chordae tendineae.

The aim of the present research is to determine the influence of the calcification of human mitral valves on the mechanical properties of their marginal chordae tendineae. The study was performed on marginal chords obtained from thirteen human mitral valves, explanted at surgery, including six non-calcified, four moderately calcified and three strongly calcified valves. The mechanical response of the chords from the non-calcified and moderately calcified valves was determined by means of quasi-static tensile tests (the poor condition of the strongly calcified valves prevented them from being mechanically characterised). The material parameters that were obtained and analysed (the Young's modulus, the secant modulus, the proportional limit stress, the ultimate strength, the strain at fracture and the density of energy stored up to maximum load) revealed noticeable differences in mechanical behaviour between the two groups of mitral chordae tendineae. Large scatter was obtained in all cases, nevertheless, considering the mean values, it was observed that the normal chords are between three and seven times stiffer or more resistant than the moderately calcified ones. On the contrary, the results obtained for the strain at fracture showed a rather different picture as, in this case, no significant differences were observed between the two families of chords. A scanning electron microscopy study was conducted in order to find out the relevant features of the calcium deposits present in the calcified chordae tendineae. In addition, the general aspects appreciated in the stress vs. strain curves were correlated with the collagen morphological evidences determined microscopically. Finally, the calcium content present in the three groups of chords was quantitatively determined through atomic absorption spectroscopy; then, the relation between the mechanical properties of normal and moderately calcified chords as a function of its calcium content was obtained. This analysis confirmed the existence of a strong correlation between calcium content and stiffness or resistance whereas the influence on the ductility seems to be negligible.

The coronary arteries of the C57BL/6 mouse strains: implications for comparison with mutant models.

There are few detailed descriptions of the coronary arterial patterns in the mouse. Some recent reports on coronary anomalies in mutant mouse models have uncovered the importance of several genes (i.e. iv and connexin43) in coronary morphogenesis. These mutations spontaneously appeared (iv) or were generated (connexin43) in a C57BL/6 background, which is widely used for the development of mutant mice. We have studied the origin and course of the main coronary arteries of two C57BL/6 mouse strains. Unusual anatomical coronary arterial patterns were found, including: solitary ostium in aorta, accessory ostium, high take-off, aortic intramural course, slit-like ostium, sinus-like ostium and origin of a septal artery from the left coronary artery. In humans, some of these conditions are clinically relevant. Most of these patterns, which differ from those observed in wild mice and Swiss albino mice, coincide with those previously found in iv/iv and connexin43 knockout mice. The results indicate that there is variability in the coronary arterial arrangement of the laboratory mouse. Care should be taken when analysing coronary phenotypes of mutant mouse models.

Basement membrane heterogeneity during chick development as shown by tomato (Lycopersicon esculentum) lectin binding.

Basement membranes (BMs) constitute a distinct compartment of the extracellular matrix (ECM). All BMs show a similar structural appearance but differ in molecular composition. These variations have critical functional implications. The aim of this study is to establish the pattern of the tomato lectin (Lycopersicon esculentum agglutinin--LEA) binding sites in the BMs of the developing chick embryo (stages 4-21, Hamburger and Hamilton, 1951) in order to achieve a better understanding of the molecular heterogeneity of BMs. The study was performed with transmission electron microscopy (TEM) histochemistry, and confocal laser microscopy. TEM showed that LEA bound to the lamina densa and to the lamina fibroreticularis of the BMs. Through the period studied, most of the LEA binding appeared in the ectodermal BM and its derivatives. In the limb bud, LEA binding to the ectoderm BM was more intense in the ventral half than in the dorsal half. Furthermore, LEA allowed the early (HH16) detection of the transverse fibrillar tracts. In the lens and in the inner ear primordium, the BMs were LEA positive through the placode and cup stages. The binding was progressively reduced through the vesicle stage. The BMs of the olfactory primordium, and of the Rathke's pouch were positive. In contrast, the BMs of the developing central nervous system were negative. The BMs of both the paraxial and the lateral plates of the mesoderm were negative, whereas the notochord and the BM of the Wolffian duct were positive. The endodermal BM and its derivatives were negative. The ECM located between the fusing endocardial tubes, and the BM of the fusion zone of the paired aortae, were positive. This suggested an active role of the LEA-positive glycoproteins in the fusion of endothelia. Our results show the heterogeneity of the chick embryo BMs during development. In addition, LEA constitutes an excellent marker for the primordial germ cells.

Cardiac morphodynamic remodelling in the growing eel (Anguilla anguilla L.).

The morphodynamic changes occurring during growth were evaluated in the eel (Anguilla anguilla L.) heart. Using an in vitro working heart preparation, cardiac performance of small (body mass 96.76 +/- 27.49 g; mean +/- s.d.) and large (body mass 656 +/- 12 g; mean +/- s.d.) eels was compared under basal conditions and under loading (i.e. preload and afterload) challenges. A parallel morphometric evaluation of the ventricle was made using light and transmission electron microscope images. The small eel hearts show a basal cardiac output lower than their large counterparts (heart rate fh, 38.93 +/- 2.82 and 52.7 +/- 1.8 beats min(-1), respectively; stroke volume Vs, 0.27 +/- 0.017 and 0.37 +/- 0.016 ml kg(-1), respectively; means +/- s.e.m.). The two groups show similar responses at increasing preload, but differ remarkably at increasing afterload. Small eel hearts decreased Vs at afterload greater than 3 kPa, in contrast to larger hearts, which maintained constant Vs up to 6 kPa. These changes in mechanical performance are related to structural differences. Compared with the small eels, the large eels show an increase in the compacta thickness and in the diameter of the trabeculae in the spongiosa, together with reduction of the lacunary spaces. The increased compacta thickness is attained by enlargements of both the muscular and vascular compartments and reduction of the interstitium; consequently, this layer appears more compacted. Both compacta and spongiosa show higher number of myocytes together with reduced cross-sectional area and myofibrillar compartment. The compacta also shows an increased mitochondrial compartment. Our results document a cardiac morphodynamic remodelling in the growing eel.

The conus valves of the adult gilthead seabream (Sparus auratus).

The conus (bulbo-ventricular) valves of teleosts perform a key function in the control of blood backflow during ventricular diastole. However, the structural characteristics of these valves are almost unknown. This paper presents a systematic anatomical, histological and structural study of the conus valves of the adult gilthead seabream (Sparus auratus). S. auratus shows two major left and right valves consisting of the leaflet and the supporting sinus. Each valvar leaflet can be divided into a stout proximal body and a flap-like distal region. The proximal body is structured into three layers: a luminal fibrosa, a dense cellular core and a parietal fibrosa. The luminal fibrosa is a collagenous structure extending the entire length of the leaflet, while the parietal fibrosa is restricted to the most proximal area. The dense cellular core consists of fibroblastic cells and a matrix rich in glycoconjugates, collagen and elastin. The histochemical and structural data suggest that the luminal fibrosa bears most of the force associated with valvar closure, while the cellular core acts as a cushion dampening vibrations and absorbing the elastic recoil. The sinus wall is a fibrous layer which shows proximal-distal differences in thickness. It also shows compositional differences that can be related to mechanical function. We describe the presence of a fibrous cylinder formed by the sinus wall, the fibrous interleaflet triangles and the fibrous layer that covers the inner surface of the conus myocardium. This fibrous cylinder constitutes the structural nexus between the ventricle, the conus and the bulbus arteriosus, provides support for the conus valves and separates the valvar complex from the surrounding tissues. The structure of the conus valves in S. auratus is different from that found in other vertebrates. Anatomical similarities between the conus valves and the mammalian arterial valves are emphasized. Each phyletic group appears to have developed specific structures in order to perform similar functions.

The conus arteriosus of the adult gilthead seabream (Sparus auratus).

This paper reports on the presence of the conus arteriosus in the heart of the adult gilthead seabream, Sparus auratus (Perciformes, Teleostei). The junctional region between the single ventricle and the bulbus arteriosus has been studied by conventional light microscopy, and by scanning and transmission electron microscopy. In addition, fluorescent phalloidin and antibodies against the muscle myosin heavy chains, laminin and collagen type IV have been used. The conus arteriosus is a distinct muscular segment interposed between the ventricle and the bulbus arteriosus. It is clearly different from the bulbus arteriosus due to its myocardial nature. It can also be distinguished from the ventricular myocardium because: (1) it has a conus shape; (2) it is formed by compact, well-vascularized myocardium; (3) it is surrounded on its inner and outer faces by fibrous layers rich in collagen and elastin; (4) it constitutes the anatomical support of the so-termed conus valves; (5) it shows intense staining for laminin and type-IV collagen; and (6) the myocardial cells located close to the inner fibrous layer are helicoidally arranged. By contrast, the ventricular myocardium is highly trabecular, lacks a compacta, shows no vessels, and presents barely detectable amounts of laminin and collagen type IV. The presence of a distinct conus arteriosus in the heart of an evolutionary advanced teleost species indicates that the conus is not a vestigial segment from the evolutionary or embryological points of view. The characteristic spatial arrangement of the conus myocytes strongly suggests that the conus is implicated in the mechanical performance of the conus valves.

Origin and course of the coronary arteries in normal mice and in iv/iv mice.

This paper reports on the origin and distribution of the coronary arteries in normal mice and in mice of the iv/iv strain, which show situs inversus and heterotaxia. The coronary arteries were studied by direct observation of the aortic sinuses with the scanning electron microscope, and by examination of vascular corrosion casts. In the normal mouse, the left and right coronaries (LC, RC) arise from the respective Valsalva sinus and course along the ventricular borders to reach the heart apex. Along this course the coronary arteries give off small branches at perpendicular or acute angles to supply the ventricles. The ventricular septum is supplied by the septal artery, which arises as a main branch from the right coronary. Conus arteries arise from the main coronary trunks, from the septal artery and/or directly from the Valsalva sinus. The vascular casts demonstrate the presence of intercoronary anastomoses. The origin of the coronary arteries was found to be abnormal in 84% of the iv/iv mice. These anomalies included double origin, high take-off, slit-like openings and the presence of a single coronary orifice. These anomalies occurred singly or in any combination, and were independent of heart situs. The septal artery originated from RC in most cases of situs solitus but originated predominantly from LC in situs inversus hearts. Except for this anomalous origin no statistical correlation was found between the coronary anomalies and heart situs or a particular mode of heterotaxia. The coronary anomalies observed in the iv/iv mice are similar to those found in human hearts. Most coronary anomalies appear to be due to defective connections between the aortic root and the developing coronaries. iv/iv mice may therefore constitute a good model to study the development of similar anomalies in the human heart.

Pitx2 expression defines a left cardiac lineage of cells: evidence for atrial and ventricular molecular isomerism in the iv/iv mice.

The homeobox gene Pitx2 has been characterized as a mediator of left-right signaling in heart, gut, and lung morphogenesis. However, the relationship between the developmental role of Pitx2 and its expression pattern at the organ level has not been explored. In this study we focus on the role of Pitx2 in heart morphogenesis. Chicken Pitx2 transcripts are present in the left portion of the cardiac crescent and in the left side of the heart tube. Through looping Pitx2 is present in the left atrium, in the ventral portion of the ventricles and in the left-ventral part of the outflow tract. Mouse Pitx2 shows a similar developmental profile of expression. To test whether Pitx2 represents a lineage marker we have tagged the left portion of the chicken cardiac tube with fluorescent DiD. Labeled cells were found at HH16 in the left atrium and in the ventral region of the ventricles and the outflow tract. In the iv/iv mouse model of cardiac heterotaxia Pitx2 was abnormally expressed in the atrial and in the ventricular chambers. Furthermore, altered Pitx2 expression correlated with the occurrence of DORV. Our data reveal the existence of molecular isomerism not only in the atrial, but also in the ventricular compartment of the heart.

Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts.

OBJECTIVES: Within the adult heart, it is convention to distinguish the conduction system and working (atrial and ventricular) myocardium. The adult conduction system (CS) comprises the sinoatrial (SAN), and atrioventricular (AVN) nodes, the atrioventricular bundle (AVB), the bundle branches and the peripheral Purkinje fibers, each of which display distinct functional properties and distinct profile of gene expression. Characterization of the mouse cardiac conduction system during development is rudimentary at present, even though genetically-modified mice are an increasing source of information regarding cardiac function and embryonic heart development. METHODS: We have performed a detailed study of the pattern of expression of myosin heavy chain (MHC), myosin light chain (MLC), troponin I (TnI) isoforms, connexin 43 (Cx43), desmin and alpha-smooth muscle actin (alpha-SMA), in the ventricular conduction system of normal and congenitally malformed mouse hearts (iv background) from embryonic day 14.5 to 19.5. RESULTS: The AVN is characterized by co-expression of MHC and MLC isoforms and no detectable expression of Cx43, desmin or alpha-SMA. The AVB expresses betaMHC and MLC2v, but no alphaMHC, MLC2a, Cx43, desmin or alpha-SMA. The right and left bundle branches display enhanced expression of desmin and alpha-SMA but no Cx43. The normal expression profile is maintained in congenitally malformed hearts such as double-outlet right ventricle and common atrioventricular canal. Three-dimensional reconstruction of the conduction system shows normal arrangement of the bundle branches in congenitally malformed hearts, but abnormal location and/or extension of the AVN. CONCLUSIONS: Molecular characterization allows to follow the development of the CS in both, normal and malformed mouse hearts. Normal phenotypic expression of the CS is independent of heart situs but shows minor modifications in the presence of heart malformations. It is concluded that the AVN derives from the atrioventricular canal myocardium, the bundle of His from the ventricular myocardium, and the bundle branches from the ventricular trabeculations. Our results do not provide evidence to support an extra-cardiac origin of the ventricular CS.

Light and electron microscopy of the bulbus arteriosus of the European eel (Anguilla anguilla).

The bulbus arteriosus of teleost fish acts as an elastic reservoir that dilates during ventricular systole to store a large part of the cardiac stroke volume. Despite its functional importance, the knowledge of the structure of the bulbus wall is still fragmentary. We have undertaken a series of studies in order to establish a general morphological plan of the teleost bulbus. The bulbus arteriosus of the European eel is studied here by means of conventional light, and transmission and scanning electron microscopy. The inner surface of the bulbus wall is irregular due to the presence of branching ridges that flatten and disappear toward the ventral aorta. The ridge surface is covered by flattened endocardial cells that show moderately dense bodies. In the ridge tissue, cells near the endocardium are mostly undifferentiated and appear isolated in a loose filamentous matrix. Ridge cells progressively cluster toward the middle layer, become surrounded by a dense matrix, and adopt characteristics typical of smooth muscle cells. This suggests the existence of a differentiation gradient. The middle layer is formed by typical smooth muscle cells embedded in a meshwork matrix that contains thin and thick filaments. Stretching of this meshwork suggests an active role of smooth muscle cells in bulbus wall dynamics. Furthermore, large areas of the extracellular space are occupied by elastin-like material. The amount of this material decreases toward the external layer. Collagen is demonstrated across the entire thickness of the bulbus wall, its amount and organization increasing from the inner toward the outer bulbus surface. The existence of matrix gradients should progressively increase wall strength, maintaining bulbus dilation within safe physiological parameters. The epicardium is formed by flattened cells that contain numerous pinocytotic vesicles, suggesting an active interchange of solutes with the pericardial cavity.

Bulbus arteriosus of the Antarctic teleosts. II. The red-blooded Trematomus bernacchii.

The structure of the bulbus arteriosus of the Antarctic teleost, Trematomus bernacchii, has been studied by light, scanning, and transmission electron microscopy. The wall of the bulbus arteriosus is divided into endocardial, subendocardial, middle and external layers. The endocardial endothelium covers the inner surface of the bulbus wall and invaginates into the subendocardium to form solid epithelial cords that show secretory activity. The subendocardial tissue is divided into finger-like ridges. Ridge cells located under the endocardium appear in niches limited by collagen fibers and thin cell extensions. Away from the endocardium ridge cells cluster into small groups, show some of the characteristics of smooth muscle cells, and appear enmeshed in a filamentous meshwork that lacks collagen and elastin fibers. The middle bulbus layer is formed by typical smooth muscle cells that are enmeshed in a filamentous meshwork similar to that observed in the ridges. The ridges and the middle layer appear to be formed by the same cell type, smooth muscle, with a gradient of differentiation from the endocardium toward the middle layer. In the absence of elastin fibers the filamentous meshwork should confer elastic properties to the bulbus wall. The stretching of the meshwork along the main axis of the middle layer cells, and between different cellular layers, suggests the existence of tensile stress and, hence, the involvement of smooth muscle cells in bulbus wall dynamics. The external layer is formed by numerous cellular types embedded in a collagenous matrix. Among these cellular types, myofibroblasts, macrophages, granulocytes, lymphocytes, dendrite-like cells, degenerating cells, and plasma cells can be recognized. The subepicardial tissue appears to be a specialized site involved in the production of the humoral immune response and displays many of the morphological characteristics of a germinal center. The outer limiting layer of the bulbus, the visceral pericardium, is formed by epithelial cells that show desmosomes and tight junctions. This suggests a close control of permeability with respect to the pericardial fluid.

Bulbus arteriosus of the antarctic teleosts. I. The white-blooded Chionodraco hamatus.

The bulbus arteriosus of teleost fish is a thick-walled chamber that extends between the single ventricle and the ventral aorta. The functional importance of the bulbus resides in the fact that it maintains a steady blood flow into the gill system through heart contraction. Despite of this, a thorough study of the structure of the bulbus in teleost fish is still lacking. We have undertaken a morphologic study of the bulbus arteriosus in the stenothermal teleosts of the Antarctic sea. The structural organization of the bulbus arteriosus of the icefish Chionodraco hamatus has been studied here by conventional light, scanning, and transmission electron microscopy. The inner surface of the bulbus shows a festooned appearance due to the presence of longitudinal, unbranched ridges that extend between the ventricle and the arterial trunk. The wall of the bulbus is divided into endocardial, subendocardial, middle, and external layers. Endocardial cells show a large number of moderately-dense bodies. The endocardium invaginates into the subendocardium forming solid epithelial cords that contain numerous secretory vacuoles. Cells in the subendocardium group into small domains, have some of the morphological characteristics of smooth muscle cells, and appear enmeshed in a three-dimensional network of matrix filaments. Cells in the middle layer are typical smooth muscle cells. They appear arranged into layers and are surrounded by a filamentous meshwork that excludes collagen fibers. Orientation of this meshwork occurs in the vicinity of the smooth muscle cells. Elastin fibers are never observed. The external layer is formed by wavy collagen bundles and fibroblast-like cells. This layer lacks blood vessels and nerve fibers. The endocardium and the endocardium-derived cords are secretory epithelia that may be involved in the formation ofmucins or glycosaminoglycans. These mucins may have a protecting effect on the endocardium. The subendocardium and the middle layer appear to be formed by the same cell type, smooth muscle, with a gradient of differentiation from the secretory (subendocardium) to the contractile (middle layer) phenotype. Despite the absence of elastin fibers, the filamentous matrix could maintain the elastic properties of the bulbus wall. Smooth muscle cells appear to be actively involved in bulbus wall dynamics. The restriction of collagen to the external layer suggests that it may control wall dilatation and bulbus compliance. When comparison was possible, structural differences between C. hamatus and temperate teleosts seemed to be not species-related, but of phenotypic adaptative significance. This is remarkable since Antarctic fishes have lived isolated in freezing waters for the last two million years.

Collagenous skeleton of the human mitral papillary muscle.

The papillary muscles (PM) of the heart have been the subject of numerous structural and functional studies. However, despite the importance of the collagenous compartment of the heart in the mechanical and electrical properties of the myocardium, little information is available on the structural organization of collagen within the PM. We study here the structural organization of collagen within the mitral papillary muscles (PM) of the human heart. Fragments of human mitral PM from normal and hypertensive subjects were macerated in NaOH to eliminate the cellular components. Macerated and nonmacerated samples were then studied with the scanning electron microscope (SEM). SEM shows that cardiac myocytes and endomysial capillaries are ensheathed in a layer of collagenous tissue. The myocyte sheath wall is formed by thin collagen fibers oriented at right angles to the main cell axis. These sheaths are open structures, collagen fibers continuing into adjacent sheaths at the points of lateral communications. Thick perimysial septa do not divide the PM tissue into separate compartments. Hypertensive hearts show perivascular and interstitial fibrosis. In addition, the lumen of the coronary vessels is reduced or obliterated, and large areas of the myocardium are substituted by densely packed collagen. Endomysial sheaths constitute a continuous collagenous layer that replicates the myocyte network. The endomysium should play a complex role in myocardial mechanics, assuring the equal distribution of force during the cardiac cycle. The absence of insulating boundaries should facilitate lateral propagation of excitation. Fibrosis in hypertensive hearts appears to be both reactive and reparative. The increase in the amount of collagen should greatly impair contractile capabilities and electrical conductance, severely compromise heart function, and contribute to development of heart failure.

Pitx2 participates in the late phase of the pathway controlling left-right asymmetry.

Pitx2, a member of the bicoid-related family of homeobox-containing genes, is asymmetrically expressed in the left lateral plate mesoderm and derived tissues during chick and mouse development. Modifications of Pitx2 pattern of expression in the iv mouse mutation correlate with the situs alterations characteristic of the mutation. Misexpression experiments demonstrate that Shh and nodal positively regulate Pitx2 expression. Our results are compatible with a Pitx2 function in the late phase of the gene cascade controlling laterality.

Developmental biology of the vertebrate heart.

This paper summarizes the development of the heart from the formation of the heart mesoderm to cardiac septation. A brief account of morphological changes is provided, but attention is focused on mechanisms rather than on morphologic descriptions. Heart induction and differentiation, and the expression of cardiac specific proteins, are reviewed. New developments in these areas include the possible role of cell surface proteins and peptide growth factors in the segregation of the splanchnic mesoderm and in cardiac commitment. Past and recent experiments indicate that the heart morphogenetic information is engraved in the precardiac mesoderm. In spite of this, specific differentiative signals can be overriden experimentally demonstrating the unstability of the cardiac phenotype at the early heart tube stage. The relationship between differentiation and morphogenesis is analyzed. While cardiac differentiation appears to be a prerequisite for morphogenesis, a number of experiments indicate that differentiation can proceed in the absence of any morphogenesis. Formation of the heart loop is separated into two different components; looping itself and the acquisition of handedness. Late heart morphogenesis is explained in terms of differential tissue growth and tissue remodeling. This not only includes morphogenetic changes intrinsic to the heart but the addition of new cell types (neural crest, epicardium, vessels, nerves) that become integrated into the developing heart. The contribution of specific mechanisms to our understanding of heart development, such as cell death and hemodynamics is also analyzed.

Atrioventricular valves of the mouse: III. Collagenous skeleton and myotendinous junction.

BACKGROUND: The leaflet tissue of the mouse atrioventricular (AV) valves contains a system of wavy collagen bundles that organize like tendons, orientate along lines of tension, and constitute an essential component of the valve tissue. The organization of these bundles is different in the two AV valves, reflecting differences in the anatomy of the entire valvular complex. Further insights into this kind of organization are needed to gain a complete understanding of the functional anatomy of the mouse AV valves. METHODS: The endocardial covering of the mouse AV valves (from 21 days to 1 year of age) was eliminated by the sonication or the maceration method. This allowed us to study in situ the organization of the collagenous valve skeleton, as well as the structure of the myotendinous junction. RESULTS: The leaflets of the two AV valves are formed by a fibrous layer (on the ventricular side) and a spongy layer (on the atrial side). The fibrosa is formed by undulating collagen bundles that organize and orientate differently on the right and left sides. The spongiosa is formed, on both sides, by a loose network of thin collagen fibers with no apparent orientation. Myocardial cells in the papillary muscles of the tricuspid valve are elongated and show cone-shaped tips. Collagen fibers attach to the myocyte surface. Collagen struts and thin septa can also be recognized. On the other hand, the collagenous components of the mitral leaflets attach tangentially to the mitral papillary muscles. On the two sides, the myocytes appear to be ensheathed in a layer of collagenous tissue. The sheaths are formed by circularly arranged fibers and appear to be tightly interconnected. CONCLUSIONS: The differences in the collagenous organization between the two AV valves reflect differences in the gross anatomy of the valves. The attachment of collagen to the papillary myocytes in the tricuspid valve resembles that of a typical myotendinous junction. However, the collagen-muscle junction in the mitral valve is more similar to the structure of a pennate muscle. The collagen matrix of the heart has been divided into endomysial, perimysial, and epimysial components. The presence of sheaths housing individual myocytes and capillaries, struts, and thin septa, corresponds to the endomysium. The absence of perimysial septa, which aggregate myocytes into groups, is striking, but this may just be a species difference. The appropriateness of the term epimysium, as applied to the heart, is discussed.

Atrioventricular valves of the mouse: II. Light and transmission electron microscopy.

BACKGROUND: Mouse atrioventricular (AV) valves present a number of conspicuous morphologic differences with human AV valves. Given the existence of these differences, it is important to know the structural organization of mouse AV valves. Since the mouse is often considered to be a good animal model for developmental and anatomical studies, the presence of significant differences in structure may render comparative studies difficult. In addition, we wished to learn about the existence of structural changes in the mouse AV valves with age. METHODS: The structural organization of mouse AV valves from 21 days to 1 year of age was studied by polarizing microscopy and by conventional light and transmission electron microscopy. RESULTS: Polarizing microscopy reveals the presence of a system of birefringent fibers that consist of collagen bundles that organize like tendons. The spatial organization of these fibers is different in the two AV valves, reflecting differences in the anatomy of the entire valvular complex. Interstitial cells (IC) are of two different phenotypes: some are typical fibroblasts, while some others share smooth muscle cell characteristics. In addition, small areas of fibrocartilage are also observed. The compactness and thickness of the collagen bundles increase with age. Also with age, the basement membranes become thickened or multilayered, and matrix vesicles and deposits of amyloid P can be observed. CONCLUSIONS: The collagenous birefringent fibers form an internal skeleton that should transmit the cycling stress evenly over the entire leaflets. IC should help to maintain the structure and deformability of the valve tissue and appear actively involved in the synthesis and renewal of extracellular material. The cartilaginous foci appear to be a normal component of the valve tissue. The structural changes observed in old animals appear to be related to the degenerative processes which take place in normal valvular tissues with age. Despite the structural differences, age changes appear to be similar in the AV valves of mouse and man.

Lectin-binding sites during postnatal differentiation of normal and cystic rabbit renal corpuscles.

Fluorochrome-labeled lectins were used to study the expression of glycoconjugates during the postnatal differentiation of normal and cystic rabbit renal corpuscles. Glomerular cysts (GC) are induced in the rabbit by a single injection of corticoids. The Bowman's capsule of these cysts is exclusively formed of podocytes (parietal podocytes). During normal development, the cell coat of the podocytes is intensely positive for wheat germ agglutinin (WGA) and Maclura pomifera agglutinin (MPA). This reaction decreases considerably during maturation, in parallel with an increase in the number of binding sites masked by terminal sialylation. Throughout the stages studied, the podocyte coat is peanut agglutinin (PNA)-negative, but it becomes intensely positive after neuraminidase treatment. Visceral and parietal podocytes in the glomerular cysts show the same pattern of glycosylation as the normal podocytes. In contrast, normal parietal cells only transiently expressed a weak reactivity to WGA and MPA during the first stages of differentiation, and did not express cryptic binding sites at any stage. The glomerular basement membrane (GBM) is positive to WGA, to succinylated WGA, and to MPA, in all the stages studied. Maturation of the GBM is characterized by expression of cryptic MPA-binding sites, and by a considerable increase in the number of cryptic PNA-binding sites. The basement membrane of the parietal layer of the cystic Bowman's capsule shows the same pattern of glycosylation, despite the fact that this epithelial layer is solely formed of podocytes and lacks endothelial cells. In contrast, the normal parietal basement membrane does not express PNA or MPA cryptic sites at any stage.(ABSTRACT TRUNCATED AT 250 WORDS)