Anguimorpha as a model group for studying the comparative heart morphology among Lepidosauria: Evolutionary window on the ventricular septation.

The group Anguimorpha represents one of the most unified squamate clades in terms of body plan, ecomorphology, ecophysiology and evolution. On the other hand, the anguimorphs vary between different habitats and ecological niches. Therefore, we focused on the group Anguimorpha to test a possible correlation between heart morphology and ecological niche with respect to phylogenetic position in Squamata with Sphenodon, Salvator, and Pogona as the outgroups. The chosen lepidosaurian species were investigated by microCT. Generally, all lepidosaurs had two well-developed atria with complete interatrial septum and one ventricle divided by ventricular septa to three different areas. The ventricles of all lepidosaurians had a compact layer and abundant trabeculae. The compact layer and trabeculae were developed in accordance with particular ecological niche of the species, the trabeculae in nocturnal animals with low metabolism, such as Sphenodon, Heloderma or Lanthanotus were more massive. On the other hand athletic animals, such as varanids or Salvator, had ventricle compartmentalization divided by three incomplete septa. A difference between varanids and Salvator was found in compact layer thickness: thicker in monitor lizards and possibly linked to their mammalian-like high blood pressure, and the level of ventricular septation. In summary: heart morphology varied among clades in connection with the ecological niche of particular species and it reflects the phylogenetic position in model clade Anguimorpha. In the absence of fossil evidence, this is the closest approach how to understand heart evolution and septation in clade with different cardiac compartmentalization levels.

Myocardial development in crocodylians.

BACKGROUND: Recent reports confirmed the notion that there exists a rudimentary cardiac conduction system (CCS) in the crocodylian heart, and development of its ventricular part is linked to septation. We thus analyzed myocardial development with the emphasis on the CCS components and vascularization in two different crocodylian species. RESULTS: Using optical mapping and HNK-1 immunostaining, pacemaker activity was localized to the right-sided sinus venosus. The atrioventricular conduction was restricted to dorsal part of the atrioventricular canal. Within the ventricle, the impulse was propagated from base-to-apex initially by the trabeculae, later by the ventricular septum, in which strands of HNK-1 positivity were temporarily observed. Completion of ventricular septation correlated with transition of ventricular epicardial activation pattern to mature apex-to-base direction from two periapical foci. Despite a gradual thickening of the ventricular wall, no morphological differentiation of the Purkinje network was observed. Thin-walled coronary vessels with endothelium positive for QH1 obtained a smooth muscle coat after septation. Intramyocardial vessels were abundant especially in the rapidly thickening left ventricular wall. CONCLUSIONS: Most of the CCS components present in the homeiotherm hearts can be identified in the developing crocodylian heart, with a notable exception of the Purkinje network distinct from the trabeculae carneae.

The Tale-Tell Heart: Evolutionary tetrapod shift from aquatic to terrestrial life-style reflected in heart changes in axolotl (Ambystoma mexicanum).

BACKGROUND: During amphibian metamorphosis, the crucial moment lies in the rearrangement of the heart, reflecting the changes in circulatory demands. However, little is known about the exact shifts linked with this rearrangement. Here, we demonstrate such myocardial changes in axolotl (Ambystoma mexicanum) from the morphological and physiological point of view. RESULTS: Micro-CT and histological analysis showed changes in ventricular trabeculae organization, completion of the atrial septum and its connection to the atrioventricular valve. Based on Myosin Heavy Chain and Smooth Muscle Actin expression we distinguished metamorphosis-induced changes in myocardial differentiation at the ventricular trabeculae and atrioventricular canal. Using optical mapping, faster speed of conduction through the atrioventricular canal was demonstrated in metamorphic animals. No differences between the groups were observed in the heart rates, ventricular activation times, and activation patterns. CONCLUSIONS: Transition from aquatic to terrestrial life-style is reflected in the heart morphology and function. Rebuilding of the axolotl heart during metamorphosis was connected with reorganization of ventricular trabeculae, completion of the atrial septum and its connection to the atrioventricular valve, and acceleration of AV conduction.

Tissue clearing and imaging methods for cardiovascular development.

Tissue imaging in 3D using visible light is limited and various clearing techniques were developed to increase imaging depth, but none provides universal solution for all tissues at all developmental stages. In this review, we focus on different tissue clearing methods for 3D imaging of heart and vasculature, based on chemical composition (solvent-based, simple immersion, hyperhydration, and hydrogel embedding techniques). We discuss in detail compatibility of various tissue clearing techniques with visualization methods: fluorescence preservation, immunohistochemistry, nuclear staining, and fluorescent dyes vascular perfusion. We also discuss myocardium visualization using autofluorescence, tissue shrinking, and expansion. Then we overview imaging methods used to study cardiovascular system and live imaging. We discuss heart and vessels segmentation methods and image analysis. The review covers the whole process of cardiovascular system 3D imaging, starting from tissue clearing and its compatibility with various visualization methods to the types of imaging methods and resulting image analysis.

Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

The formation of the atrioventricular conduction axis is linked in development to ventricular septation.

During development, the ventricles of mammals and birds acquire a specialized pattern of electrical activation with the formation of the atrioventricular conduction system (AVCS), which coincides with the completion of ventricular septation. We investigated whether AVCS formation coincides with ventricular septation in developing Siamese crocodiles (Crocodylus siamensis). Comparisons were made with Amazon toadhead turtle (Mesoclemmys heliostemma) with a partial septum only and no AVCS (negative control) and with chicken (Gallus gallus) (septum and AVCS, positive control). Optical mapping of the electrical impulse in the crocodile and chicken showed a similar developmental specialization that coincided with full ventricular septation, whereas in the turtle the ventricular activation remained primitive. Co-localization of neural marker human natural killer-1 (HNK-1) and cardiomyocyte marker anti-myosin heavy chain (MF20) identified the AVCS on top of the ventricular septum in the crocodile and chicken only. AVCS formation is correlated with ventricular septation in both evolution and development.

Developmental mechanisms driving complex tooth shape in reptiles.

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

Low incidence of atrial septal defects in nonmammalian vertebrates.

The atrial septum enables efficient oxygen transport by separating the systemic and pulmonary venous blood returning to the heart. Only in placental mammals will the atrial septum form by the coming-together of the septum primum and the septum secundum. In up to one of four placental mammals, this complex morphogenesis is incomplete and yields patent foramen ovale. The incidence of incomplete atrial septum is unknown for groups with the septum primum only, such as birds and reptiles. We found a low incidence of incomplete atrial septum in 11 species of bird (0% of specimens) and 13 species of reptiles (3% of specimens). In reptiles, there was a trabecular interface between the atrial septum and the atrial epicardium which was without a clear boundary between left and right atrial cavities. In developing reptiles (four squamates and one crocodylian), the septum primum initiated as a sheet that acquired perforations and the trabecular interface developed late. We conclude that atrial septation from the septum primum only results in a low incidence of incompleteness. In reptiles, the atrial septum and atrial wall develop a trabecular interface, but previous studies on atrial hemodynamics suggest this interface has a very limited capacity for shunting.

Identification of the building blocks of ventricular septation in monitor lizards (Varanidae).

Among lizards, only monitor lizards (Varanidae) have a functionally divided cardiac ventricle. The division results from the combined function of three partial septa, which may be homologous to the ventricular septum of mammals and archosaurs. We show in developing monitors that two septa, the 'muscular ridge' and 'bulbuslamelle', express the evolutionarily conserved transcription factors Tbx5, Irx1 and Irx2, orthologues of which mark the mammalian ventricular septum. Compaction of embryonic trabeculae contributes to the formation of these septa. The septa are positioned, however, to the right of the atrioventricular junction and they do not participate in the separation of incoming atrial blood streams. That separation is accomplished by the 'vertical septum', which expresses Tbx3 and Tbx5 and orchestrates the formation of the electrical conduction axis embedded in the ventricular septum. These expression patterns are more pronounced in monitors than in other lizards, and are associated with a deep electrical activation near the vertical septum, in contrast to the primitive base-to-apex activation of other lizards. We conclude that evolutionarily conserved transcriptional programmes may underlie the formation of the ventricular septa of monitors.

Epoxyeicosatrienoic acid analog EET-B attenuates post-myocardial infarction remodeling in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) and their synthetic analogs have cardiovascular protective effects. Here, we investigated the action of a novel EET analog EET-B on the progression of post-myocardial infarction (MI) heart failure in spontaneously hypertensive rats (SHR). Adult male SHR were divided into vehicle- and EET-B (10 mg/kg/day; p.o., 9 weeks)-treated groups. After 2 weeks of treatment, rats were subjected to 30-min left coronary artery occlusion or sham operation. Systolic blood pressure (SBP) and echocardiography (ECHO) measurements were performed at the beginning of study, 4 days before, and 7 weeks after MI. At the end of the study, tissue samples were collected for histological and biochemical analyses. We demonstrated that EET-B treatment did not affect blood pressure and cardiac parameters in SHR prior to MI. Fractional shortening (FS) was decreased to 18.4 ± 1.0% in vehicle-treated MI rats compared with corresponding sham (30.6 ± 1.0%) 7 weeks following MI induction. In infarcted SHR hearts, EET-B treatment improved FS (23.7 ± 0.7%), markedly increased heme oxygenase-1 (HO-1) immunopositivity in cardiomyocytes and reduced cardiac inflammation and fibrosis (by 13 and 19%, respectively). In conclusion, these findings suggest that EET analog EET-B has beneficial therapeutic actions to reduce cardiac remodeling in SHR subjected to MI.

HNK-1 in Morphological Study of Development of the Cardiac Conduction System in Selected Groups of Sauropsida.

Human natural killer (HNK)-1 antibody is an established marker of developing cardiac conduction system (CCS) in birds and mammals. In our search for the evolutionary origin of the CCS, we tested this antibody in a variety of sauropsid species (Crocodylus niloticus, Varanus indicus, Pogona vitticeps, Pantherophis guttatus, Eublepharis macularius, Gallus gallus, and Coturnix japonica). Hearts of different species were collected at various stages of embryonic development and studied to map immunoreactivity in cardiac tissues. We performed detection on alternating serial paraffin sections using immunohistochemistry for smooth muscle actin or sarcomeric actin as myocardial markers, and HNK-1 to visualize overall staining pattern and then positivity in specific myocyte populations. We observed HNK-1 expression of various intensity distributed in the extracellular matrix and mesenchymal cell surface of cardiac cushions in most of the examined hearts. Strong staining was found in the cardiac nerve fibers and ganglia in all species. The myocardium of the sinus venosus and the atrioventricular canal exhibited transitory patterns of expression. In the Pogona and Crocodylus hearts, as well as in the Gallus and Coturnix ones, additional expression was detected in a subset of myocytes of the (inter)ventricular septum. These results support the use of HNK-1 as a conserved marker of the CCS and suggest that there is a rudimentary CCS present in developing reptilian hearts. Anat Rec, 302:69-82, 2019. © 2018 Wiley Periodicals, Inc.

Relative position of the atrioventricular canal determines the electrical activation of developing reptile ventricles.

Squamate reptiles appear to lack the specialized His-Purkinje system that enables the cardiac ventricle to be activated from apex to base as in mammals and birds. Instead, activation may simply spread from where the atrioventricular canal connects to the base. Gja5, which encodes Cx40, which allows fast impulse propagation, was expressed throughout the ventricles of developing anole lizards. Activation was optically recorded in developing corn snake and central bearded dragon. Early embryonic ventricles were broad in shape, and activation propagated from the base to the right. Elongated ventricles of later stages were activated from base to apex. Before hatching of the snake, the ventricle developed a cranial extension on the left and activation propagated from the base to the caudal apex and the cranial extension. In squamate reptiles, the pattern of electrical activation of the cardiac ventricle is dependent on the position of the atrioventricular canal and the shape of the ventricle.

Ossification Pattern in Forelimbs of the Siamese Crocodile (Crocodylus siamensis): Similarity in Ontogeny of Carpus Among Crocodylian Species.

Crocodylians have highly derived elongated carpus, which is related to their use of forelimbs in many types of gaits as well as in burrowing. The objective of present study was to describe the ossification of the forelimb in five stages of Siamese crocodile (Crocodylus siamensis). The ossification begins approximately at stage 20 in arm and forearm bones moving sequentially to the metacarpal elements. The first carpal elements with ossification centers are radiale + intermedium and ulnare (stage 22-23), and their ossification mode is typical of long bones. Between stages 22 and 24 distal carpals 3, 4, and 5 fuse together to a single formation. In the stage 25, the ossification proceeds to the pisiform, which starts ossifying late during the embryogenesis. The phalangeal formula of the digits is 2,3,4,5,3. Although there are some interspecific differences, it appears that all crocodylians have similarly uniform skeletal pattern, the process of ossification, number of carpal elements and phalangeal formulas probably due to their similar lifestyles. Anat Rec, 301:1159-1168, 2018. © 2018 Wiley Periodicals, Inc.

Reactions of green lizards (Lacerta viridis) to major repellent compounds secreted by Graphosoma lineatum (Heteroptera: Pentatomidae).

The chemical defence of Heteroptera is primarily based on repellent secretions which signal the potential toxicity of the bug to its predators. We tested the aversive reactions of green lizards (Lacerta viridis) towards the major compounds of the defensive secretion of Graphosoma lineatum, specifically: (i) a mixture of three aldehydes: (E)-hex-2-enal, (E)-oct-2-enal, (E)-dec-2-enal; (ii) a mixture of these three aldehydes and tridecane; (iii) oxoaldehyde: (E)-4-oxohex-2-enal; (iv) secretion extracted from metathoracic scent glands of G. lineatum adults and (v) hexane as a non-polar solvent. All chemicals were presented on a palatable food (Tenebrio molitor larvae). The aversive reactions of the green lizards towards the mealworms were evaluated by observing the approach latencies, attack latencies and approach-attack intervals. The green lizards exhibited a strong aversive reaction to the mixture of three aldehydes. Tridecane reduced the aversive reaction to the aldehyde mixture. Oxoaldehyde caused the weakest, but still significant, aversive reaction. The secretion from whole metathoracic scent glands also clearly had an aversive effect on the green lizards. Moreover, when a living specimen of G. lineatum or Pyrrhocoris apterus (another aposematic red-and-black prey) was presented to the green lizards before the trials with the aldehyde mixture, the aversive effect of the mixture was enhanced. In conclusion, the mixture of three aldehydes had the strong aversive effect and could signal the potential toxicity of G. lineatum to the green lizards.