The Elusive Hypertrophy of the Python Heart.

The Burmese python, one of the world's largest snakes, has reached celebrity status for its dramatic physiological responses associated with digestion of enormous meals. The meals elicit a rapid gain of mass and function of most visceral organs, particularly the small intestine. There is also a manyfold elevation of oxygen consumption that demands the heart to deliver more oxygen. It therefore made intuitive sense when it was reported that the postprandial response entailed a 40% growth of heart mass that could accommodate a rise in stroke volume. Many studies, however, have not been able to reproduce the 40% growth of the heart. We collated published values on postprandial heart mass in pythons, which include several instances of no change in heart mass. On average, the heart mass is only 15% greater. The changes in heart mass did not correlate to the mass gain of the small intestine or peak oxygen consumption. Hemodynamic studies show that the rise in cardiac output does not require increased heart mass but can be fully explained by augmented cardiac filling and postprandial tachycardia. Under the assumption that hypertrophy is a contingent phenomenon, more recent experiments have employed two interventions such as feeding with a concomitant reduction in hematocrit. The results suggest that the postprandial response of the heart can be enhanced, but the 40% hypertrophy of the python heart remains elusive.

The changing morphology of the ventricular walls of mouse and human with increasing gestation.

That the highly trabeculated ventricular walls of the developing embryos transform to the arrangement during the fetal stages, when the mural architecture is dominated by the thickness of the compact myocardium, has been explained by the coalescence of trabeculations, often erroneously described as 'compaction'. Recent data, however, support differential rates of growth of the trabecular and compact layers as the major driver of change. Here, these processes were assessed quantitatively and visualized in standardized views. We used a larger dataset than has previously been available of mouse hearts, covering the period from embryonic day 10.5 to postnatal day 3, supported by images from human hearts. The volume of the trabecular layer increased throughout development, in contrast to what would be expected had there been 'compaction'. During the transition from embryonic to fetal life, the rapid growth of the compact layer diminished the proportion of trabeculations. Similarly, great expansion of the central cavity reduced the proportion of the total cavity made up of intertrabecular recesses. Illustrations of the hearts with the median value of left ventricular trabeculation confirm a pronounced growth of the compact wall, with prominence of the central cavity. This corresponds, in morphological terms, to a reduction in the extent of the trabecular layer. Similar observations were made in the human hearts. We conclude that it is a period of comparatively slow growth of the trabecular layer, rather than so-called compaction, that is the major determinant of the changing morphology of the ventricular walls of both mouse and human hearts.

Evolutionary Aspects of Chamber Formation and Septation.

The formed hearts of vertebrates are widely different in anatomy and performance, yet their embryonic hearts are surprisingly similar. Developmental and molecular biology are making great advances in reconciling these differences by revealing an evolutionarily conserved building plan to the vertebrate heart. This suggests that perspectives from evolution may improve our understanding of the formation of the human heart. Here, we exemplify this approach by discussing atrial and ventricular septation and the associated processes of remodeling of the atrioventricular junction and formation of the atrioventricular insulating plane.

Molecular Pathways and Animal Models of Ebstein's Anomaly.

Ebstein's anomaly is a congenital malformation of the tricuspid valve characterized by abnormal attachment of the valve leaflets, resulting in varying degrees of valve dysfunction. The anatomic hallmarks of this entity are the downward displacement of the attachment of the septal and posterior leaflets of the tricuspid valve. Additional intracardiac malformations are common. From an embryological point of view, the cavity of the future right atrium does not have a direct orifice connected to the developing right ventricle. This chapter provides an overview of current insight into how this connection is formed and how malformations of the tricuspid valve arise from dysregulation of molecular and morphological events involved in this process. Furthermore, mouse models that show features of Ebstein's anomaly and the naturally occurring model of canine tricuspid valve malformation are described and compared to the human model. Although Ebstein's anomaly remains one of the least understood cardiac malformations to date, the studies summarized here provide, in aggregate, evidence for monogenic and oligogenic factors driving pathogenesis.

Trabeculations of the porcine and human cardiac ventricles are different in number but similar in total volume.

An intricate meshwork of trabeculations lines the luminal side of cardiac ventricles. Compaction, a developmental process, is thought to reduce trabeculations by adding them to the neighboring compact wall which is then enlarged. When pig, a plausible cardiac donor for xenotransplantation, is compared to human, the ventricular walls appear to have fewer trabeculations. We hypothesized the trabecular volume is proportionally smaller in pig than in human. Macroscopically, we observed in 16 pig hearts that the ventricular walls harbor few but large trabeculations. Close inspection revealed a high number of tiny trabeculations, a few hundred, within the recesses of the large trabeculations. While tiny, these were still larger than embryonic trabeculations and even when considering their number, the total tally of trabeculations in pig was much fewer than in human. Volumetrics based on high-resolution MRI of additional six pig hearts compared to six human hearts, revealed the left ventricles were not significantly differently trabeculated (21.5 versus 22.8%, respectively), and the porcine right ventricles were only slightly less trabeculated (42.1 vs 49.3%, respectively). We then analyzed volumetrically 10 pig embryonic hearts from gestational day 14-35. The trabecular and compact layer always grew, as did the intertrabecular recesses, in contrast to what compaction predicts. The proportions of the trabecular and compact layers changed substantially, nonetheless, due to differences in their growth rate rather than compaction. In conclusion, processes that affect the trabecular morphology do not necessarily affect the proportion of trabecular-to-compact myocardium and they are then distinct from compaction.

Laterality defect of the heart in non-teleost fish.

Dextrocardia is a rare congenital malformation in humans in which most of the heart mass is positioned in the right hemithorax rather than on the left. The heart itself may be normal and dextrocardia is sometimes diagnosed during non-related explorations. A few reports have documented atypical positions of the cardiac chambers in farmed teleost fish. Here, we report the casual finding of a left-right mirrored heart in an 85 cm long wild-caught spiny dogfish (Squalus acanthias) with several organ malformations. Macroscopic observations showed an outflow tract originating from the left side of the ventricular mass, rather than from the right. Internal inspection revealed the expected structures and a looped cavity. The inner curvature of the loop comprised a large trabeculation, the bulboventricular fold, as expected. The junction between the sinus venosus and the atrium appeared normal, only mirrored. MRI data acquired at 0.7 mm isotropic resolution and subsequent 3D-modeling revealed the atrioventricular canal was to the right of the bulboventricular fold, rather than on the left. Spurred by the finding of dextrocardia in the shark, we revisit our previously published material on farmed Adriatic sturgeon (Acipenser naccarii), a non-teleost bony fish. We found several alevins with inverted (left-loop) hearts, amounting to an approximate incidence of 1%-2%. Additionally, an adult sturgeon measuring 90 cm in length showed abnormal topology of the cardiac chambers, but normal position of the abdominal organs. In conclusion, left-right mirrored hearts, a setting that resembles human dextrocardia, can occur in both farmed and wild non-teleost fish.

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.

Anatomy of the heart with the highest heart rate.

Shrews occupy the lower extreme of the seven orders of magnitude mammals range in size. Their hearts are large relative to body weight and heart rate can exceed a thousand beats a minute. It is not known whether traits typical of mammal hearts scale to these extremes. We assessed the heart of three species of shrew (genus Sorex) following the sequential segmental analysis developed for human hearts. Using micro-computed tomography, we describe the overall structure and find, in agreement with previous studies, a large and elongate ventricle. The atrial and ventricular septums and the atrioventricular (AV) and arterial valves are typically mammalian. The ventricular walls comprise mostly compact myocardium and especially the right ventricle has few trabeculations on the luminal side. A developmental process of compaction is thought to reduce trabeculations in mammals, but in embryonic shrews the volume of trabeculations increase for every gestational stage, only slower than the compact volume. By expression of Hcn4, we identify a sinus node and an AV conduction axis which is continuous with the ventricular septal crest. Outstanding traits include pulmonary venous sleeve myocardium that reaches farther into the lungs than in any other mammals. Typical proportions of coronary arteries-to-aorta do not scale and the shrew coronary arteries are proportionally enormous, presumably to avoid the high resistance to blood flow of narrow vessels. In conclusion, most cardiac traits do scale to the miniscule shrews. The shrew heart, nevertheless, stands out by its relative size, elongation, proportionally large coronary vessels, and extent of pulmonary venous myocardium.

Anatomy of the heart of the leatherback turtle.

Non-crocodylian reptiles have hearts with a single ventricle, which is partially separated by a muscular ridge that provides some separation of blood flows. An exceptional situation exists in monitor lizards and pythons, where the ventricular left side generates a much higher systolic blood pressure than the right side, thus resembling mammals and birds. This functional division of the ventricle depends on a large muscular ridge and may relate to high metabolic demand. The large leatherback turtle (<1000 kg), with its extensive migrations and elevated body temperatures, may have similar adaptations. We report on the anatomy of the hearts of two leatherback turtles. One stranded in Ballum, Denmark in 2020, and was examined in detail, supplemented by observations and photos of an additional stranding specimen from Canada. The external morphology of the leatherback heart resembles that of other turtles, but it is large. We made morphometric measurements of the Ballum heart and created an interactive 3D model using high-resolution MRI. The volume of the ventricle was 950 ml, from a turtle of 300 kg, which is proportionally almost twice as large as in other reptiles. The Ballum heart was compared to MRI scans of the hearts of a tortoise, a python, and a monitor lizard. Internally, the leatherback heart is typical of non-crocodylian reptiles and did not contain the well-developed septation found in pythons and monitor lizards. We conclude that if leatherback turtles have exceptional circulation needs, they are sustained with a relatively large but otherwise typical non-crocodylian reptile heart.

Higher spatial resolution improves the interpretation of the extent of ventricular trabeculation.

The ventricular walls of the human heart comprise an outer compact layer and an inner trabecular layer. In the context of an increased pre-test probability, diagnosis left ventricular noncompaction cardiomyopathy is given when the left ventricle is excessively trabeculated in volume (trabecular vol >25% of total LV wall volume) or thickness (trabecular/compact (T/C) >2.3). Here, we investigated whether higher spatial resolution affects the detection of trabeculation and thus the assessment of normal and excessively trabeculated wall morphology. First, we screened left ventricles in 1112 post-natal autopsy hearts. We identified five excessively trabeculated hearts and this low prevalence of excessive trabeculation is in agreement with pathology reports but contrasts the prevalence of approximately 10% of the population found by in vivo non-invasive imaging. Using macroscopy, histology and low- and high-resolution MRI, the five excessively trabeculated hearts were compared with six normal hearts and seven abnormally trabeculated and excessive trabeculation-negative hearts. Some abnormally trabeculated hearts could be considered excessively trabeculated macroscopically because of a trabecular outflow or an excessive number of trabeculations, but they were excessive trabeculation-negative when assessed with MRI-based measurements (T/C <2.3 and vol <25%). The number of detected trabeculations and T/C ratio were positively correlated with higher spatial resolution. Using measurements on high resolution MRI and with histological validation, we could not replicate the correlation between trabeculations of the left and right ventricle that has been previously reported. In conclusion, higher spatial resolution may affect the sensitivity of diagnostic measurements and in addition could allow for novel measurements such as counting of trabeculations.

Fetal Tricuspid Valve Agenesis/Atresia: Testing Predictions of the Embryonic Etiology.

Tricuspid valve agenesis/atresia (TVA) is a congenital cardiac malformation where the tricuspid valve is not formed. It is hypothesized that TVA results from a failure of the normal rightward expansion of the atrioventricular canal (AVC). We tested predictions of this hypothesis by morphometric analyses of the AVC in fetal hearts. We used high-resolution MRI and ultrasonography on a post-mortem fetal heart with TVA and with tricuspid valve stenosis (TVS) to validate the position of measurement landmarks that were to be applied to clinical echocardiograms. This revealed a much deeper right atrioventricular sulcus in TVA than in TVS. Subsequently, serial echocardiograms of in utero fetuses between 12 and 38 weeks of gestation were included (n = 23 TVA, n = 16 TVS, and n = 74 controls) to establish changes in AVC width and ventricular dimensions over time. Ventricular length and width and estimated fetal weight all increased significantly with age, irrespective of diagnosis. Heart rate did not differ between groups. However, in the second trimester, in TVA, the ratio of AVC to ventricular width was significantly lower compared to TVS and controls. This finding supports the hypothesis that TVA is due to a failed rightward expansion of the AVC. Notably, we found in the third trimester that the AVC to ventricular width normalized in TVA fetuses as their mitral valve area was greater than in controls. Hence, TVA associates with a quantifiable under-development of the AVC. This under-development is obscured in the third trimester, likely because of adaptational growth that allows for increased stroke volume of the left ventricle.

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.

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.

Surface-Controlled Crystal Alignment of Naphthyl End-Capped Oligothiophene on Graphene: Thin-Film Growth Studied by in Situ X-ray Diffraction.

We report on the microstructure, morphology, and growth of 5,5'-bis(naphth-2-yl)-2,2'-bithiophene (NaT2) thin films deposited on graphene, characterized by grazing incidence X-ray diffraction (GIXRD) and complemented by atomic force microscopy (AFM) measurements. NaT2 is deposited on two types of graphene surfaces: custom-made samples where chemical vapor deposition (CVD)-grown graphene layers are transferred onto a Si/SiO2 substrate by us and common commercially transferred CVD graphene on Si/SiO2. Pristine Si/SiO2 substrates are used as a reference. The NaT2 crystal structure and orientation depend strongly on the underlying surface, with the molecules predominantly lying down on the graphene surface (face-on orientation) and standing nearly out-of-plane (edge-on orientation) on the Si/SiO2 reference surface. Post growth GIXRD and AFM measurements reveal that the crystalline structure and grain morphology differ depending on whether there is polymer residue left on the graphene surface. In situ GIXRD measurements show that the thickness dependence of the intensity of the (111) reflection from the crystalline edge-on phase does not intersect zero at the beginning of the deposition process, suggesting that an initial wetting layer, corresponding to 1-2 molecular layers, is formed at the surface-film interface. By contrast, the (111) reflection intensity from the crystalline face-on phase grows at a constant rate as a function of film thickness during the entire deposition.

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.

Sinus venosus incorporation: contentious issues and operational criteria for developmental and evolutionary studies.

The sinus venosus is a cardiac chamber upstream of the right atrium that harbours the dominant cardiac pacemaker. During human heart development, the sinus venosus becomes incorporated into the right atrium. However, from the literature it is not possible to deduce the characteristics and importance of this process of incorporation, due to inconsistent terminology and definitions in the description of multiple lines of evidence. We reviewed the literature regarding the incorporation of the sinus venosus and included novel electrophysiological data. Most mammals that have an incorporated sinus venosus show a loss of a functional valve guard of the superior caval vein together with a loss of the electrical sinuatrial delay between the sinus venosus and the right atrium. However, these processes are not necessarily intertwined and in a few species only the sinuatrial delay may be lost. Sinus venosus incorporation can be characterised as the loss of the sinuatrial delay of which the anatomical and molecular underpinnings are not yet understood.

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.

The hypertrabeculated (noncompacted) left ventricle is different from the ventricle of embryos and ectothermic vertebrates.

Ventricular hypertrabeculation (noncompaction) is a poorly characterized condition associated with heart failure. The condition is widely assumed to be the retention of the trabeculated ventricular design of the embryo and ectothermic (cold-blooded) vertebrates. This assumption appears simplistic and counterfactual. Here, we measured a set of anatomical parameters in hypertrabeculation in man and in the ventricles of embryos and animals. We compared humans with left ventricular hypertrabeculation (N=21) with humans with structurally normal left ventricles (N=54). We measured ejection fraction and ventricular trabeculation using cardiovascular MRI. Ventricular trabeculation was further measured in series of embryonic human and 9 animal species, and in hearts of 15 adult animal species using MRI, CT, or histology. In human, hypertrabeculated left ventricles were significantly different from structurally normal left ventricles by all structural measures and ejection fraction. They were far less trabeculated than human embryonic hearts (15-40% trabeculated volume versus 55-80%). Early in development all vertebrate embryos acquired a ventricle with approximately 80% trabeculations, but only ectotherms retained the 80% trabeculation throughout development. Endothermic (warm-blooded) animals including human slowly matured in fetal and postnatal stages towards ventricles with little trabeculations, generally less than 30%. Further, the trabeculations of all embryos and adult ectotherms were very thin, less than 50 µm wide, whereas the trabeculations in adult endotherms and in the setting of hypertrabeculation were wider by orders of magnitude. It is concluded in contrast to a prevailing assumption, the hypertrabeculated left ventricle is not like the ventricle of the embryo or of adult ectotherms. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.