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The IgCAM coxsackie–adenovirus receptor (CAR) is essential for embryonic heart development and electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analyzed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wild type and CAR knockout (KO) embryos. Surprisingly, in the absence of the CAR, cultured cardiomyocytes showed increased frequencies of beating and calcium cycling. Increased beatings of heart organ cultures were also induced by the application of reagents that bind to the extracellular region of the CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed size increases but decreased in the total numbers of membrane-localized Cx43 clusters. This was accompanied by improved cell–cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that the CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions.

Phenytoin is a known human teratogen with unknown etiology. Several mechanisms have been proposed including disturbances in folate metabolism, induction of embryonic hypoxia following phenytoin-induced bradycardia, free radical formation following re-oxygenation and phenytoin-induced maternal hyperglycemia. Using high frequency ultrasound, we demonstrated that phenytoin induced a dramatic decrease in the heart rate of embryos. This coincided with a moderate transient decrease in maternal heart rate and blood glucose levels. Embryonic heart rate had not fully recovered 24 h later in some embryos despite normal maternal physiological parameters. In a separate study, extent of hypoxia was measured using the marker pimonidazole. Phenytoin-exposed embryos did not demonstrate increased hypoxia compared to control embryos at 2, 4, 8 or 24 h dosing. Together our results show that phenytoin induces malformations as a result of a combination of insults: embryonic bradycardia, maternal bradycardia and maternal hyperglycemia. However, this does not appear to result in measurable embryonic hypoxia in our animal model.

In the embryonic heart development of mammals and birds, a straight initial heart tube undergoes left-handed helical looping, which is a remarkable and puzzling event. We are interested in the mechanism of this chiral helical looping. Recently, observations were reported that myocardial cells in the embryonic chick heart show intrinsic chirality of rotation. The chirality of myocardial cells, via anisotropic polarization of Golgi inside the cells, leads to a left-right (LR) asymmetry of cell shape. On cell boundaries of LR asymmetric cells, phosphorylated myosin and N-cadherin are enriched. Such LR asymmetric cellular circumstances lead to a large-scale three-dimensional chiral structure, the left-handed helical loop. However, the physical mechanism of this looping is unclear. Computer simulations were performed using a cell-based three-dimensional mathematical model assuming an anterior-rightward-biased contractile force of the cell boundaries on the ventral surface of the heart (orientation of a clock hand pointing to 10 to 11 o'clock). An initially straight heart tube was successfully remodeled to the left-handed helical tube via frequent convergent extension (CE) of collective cells, which corresponds to the previously reported observations of chick heart development. Although we assumed that the biased boundary contractile force was uniform all over the ventral side, orientations of the CEs became position specific on the anterior, posterior, right, and left regions on the ventral tube. Such position-specific CEs produced the left-handed helical loop. In addition, our results suggest the loop formation process consists of two distinct phases of preparation and explicit looping. Intrinsic cell properties of chirality in this investigation were discussed relating to extrinsic factors investigated by other researches. Finally, because CE is generally exerted in the axial developmental process across different animal species, we discussed the contribution of CE to the chiral heart structure across species of chick, mouse, Xenopus, and zebrafish.

During development, the heart begins pumping as a valveless multilayered tube capable of driving blood flow throughout the embryonic vasculature. The mechanical properties and how they interface with pumping function are not well-defined at this stage. Here, we evaluate pumping patterns using a fluid–structure interaction computational model, combined with experimental data and an energetic analysis to investigate myocardial mechanical properties. Through this work, we propose that a myocardium modeled as a Neo-Hookean material with a material constant on the order of 10 kPa is necessary for the heart tube to function with an optimal pressure and cardiac output.

Background Our recent epidemiological study revealed that maternal sleep during the periconceptional period should be involved in the risk of congenital heart disease (CHD) in offspring. Melatonin, a sleep related hormone, has been suggested to play a crucial role in embryonic development based on the emerging evidence. In this study, we set out to assess the effect of melatonin on the embryonic cardiac cell growth and to explore the underlying mechanisms. Methods We observed the effect of different gradient doses of melatonin as 10, 100, or 1,000 μM on cell proliferation in H9c2 embryonic rat cardiac cells. Furthermore, flow cytometry was applied to evaluate the impact on apoptosis and cell cycle. RNA-seq was conducted to screen the changes in expression of mRNA and signaling pathways. Quantitative Real-Time-PCR (qRT-PCR) was then conducted to validate the results. Results It was observed that melatonin could inhibit H9c2 cell growth, at the doses of 100 and 1,000 μM, but not at 10 μM. Moreover, melatonin ranged from 100 to 1,000 μM could instigate cell cycle arrest at G1 phase and simulate apoptosis, in a dose-dependent manner. In addition, melatonin was found to down-regulate the expression of a number of genes, which are related to heart development (SPARC, IFITM3, TNNT2, LOX), and PI3K-Akt signaling pathway activation (FN1, HSP90B1, THBS1, MFGE8, and CLU). Conclusions Our findings suggested that high level of melatonin could be capable of inhibiting growth through the induction of apoptosis and cell cycle arrest via PI3K-AKT signaling pathway, thereby interfering with embryonic heart development. Considering this study is based on H9c2 embryonic rat cardiac cells, future additional studies using human embryonic cardiac cell are warranted.

The adult mammalian heart is non-regenerative because cardiomyocytes withdraw from the cell cycle shortly after birth. Embryonic mammalian hearts, in which cardiomyocytes are genetically ablated in a salt-and-pepper-like pattern, regenerate due to compensation by residual cardiomyocytes. To date, it remains unknown whether or how transmural ventricular defects at the looped heart stage regenerate after cryoinjury. We established a cryoablation model in stage 16 chick embryonic hearts. In hearts at 5 h post cryoinjury (hpc), cryoinjury-induced defects were approximately 200 µm in width in the primitive ventricle; thereafter, the defect was filled with mesenchymal cells accumulating between the epicardium and endocardium. The defect began to regress at 4 days post cryoinjury (dpc) and disappeared around 9 dpc. Immunohistochemistry showed that there were no isl1-positive cells in either the scar tissue or residual cardiomyocytes. BrdU incorporation into residual cardiomyocytes was transiently downregulated in association with upregulation of p27 (Kip1), suggesting that cell cycle arrest occurred at G1-to-S transition immediately after cryoinjury. Estimated cell cycle length was examined, and the results showed that the shortest cell cycle length was 18 h at stages 19-23; it increased with development due to elongation of the G2-M-G1 phase and 30 h at stages 27-29. The S phase length was constant at 6-8 h. The cell cycle length was elongated immediately after cryoinjury, and it reversed at 1-2 dpc. Cryoablated transmural defects in the early embryonic heart were restored by compensation by residual myocytes.

Before septation processes shape its four chambers, the embryonic heart is a straight tube that spontaneously bends and twists breaking the left-right symmetry. In particular, the heart tube is subjected to a cell remodelling inducing ventral bending and dextral torsion during the c-looping phase. In this work we propose a morphomechanical model for the torsion of the heart tube, that behaves as a nonlinear elastic body. We hypothesize that this spontaneous looping can be modeled as a mechanical instability due to accumulation of residual stresses induced by the geometrical frustration of tissue remodelling, which mimics the cellular rearrangement within the heart tube. Thus, we perform a linear stability analysis of the resulting nonlinear elastic boundary value problem to determine the onset of c-looping as a function of the aspect ratios of the tube and of the internal remodelling rate. We perform numerical simulations to study the fully nonlinear morphological transition, showing that the soft tube develops a realistic self-contacting looped shape in the physiological range of geometrical parameters.

The coxsackie-adenovirus receptor (CAR) is a cell surface transmembrane protein originally recognized for its role as a binding site for coxsackie- and adeno-viruses. As such, it is believed to play an important role in pathogenesis of myocarditis. Other studies have suggested that CAR also plays an important role in embryonic development, which is not surprising given the strong expression of the receptor in heart, brain, liver, pancreas, kidney, small intestine, and various epithelia during development. A number of studies have looked at downregulation and upregulation of CAR and have confirmed the central role of CAR during critical periods of development. These studies all demonstrated embryonic lethality with variable phenotypes: electrophysiological abnormalities, cardiac structural deformations, and extracardiac abnormalities, such as lymphatic malformations. The purpose of this review is to summarize the existing literature about CAR and formulate some questions for future studies, with an emphasis on the role of CAR during embryonic heart development.

ACE2 was observed as the cell surface receptor of the SARS-CoV-2 virus. Interestingly, we also found ACE2 positivity inside the cell nucleus. The ACE2 levels changed during cell differentiation and aging and varied in distinct cell types. We observed ACE2 depletion in the aortas of aging female mice, similarly, the aging caused ACE2 decrease in the kidneys. Compared with that in the heart, brain and kidneys, the ACE2 level was the lowest in the mouse lungs. In mice exposed to nicotine, ACE2 was not changed in olfactory bulbs but in the lungs, ACE2 was upregulated in females and downregulated in males. These observations indicate the distinct gender-dependent properties of ACE2. Differentiation into enterocytes, and cardiomyocytes, caused ACE2 depletion. The cardiomyogenesis was accompanied by renin upregulation, delayed in HDAC1-depleted cells. In contrast, vitamin D2 decreased the renin level while ACE2 was upregulated. Together, the ACE2 level is high in non-differentiated cells. This protein is more abundant in the tissues of mouse embryos and young mice in comparison with older animals. Mostly, downregulation of ACE2 is accompanied by renin upregulation. Thus, the pathophysiology of COVID-19 disease should be further studied not only by considering the ACE2 level but also the whole renin-angiotensin system.

Background: Embryonic health assessment prior to delivery is not a priority in Africa due to the lack of efficient platforms for biomarker screening of defective heart or metabolic syndromes. Beside genetic mutations, some environmental, nutritional, or epigenetic events can induce abnormal protein expression impacting embryonic heart and gut developments. Among these proteins is the retinoic acid (vitamin A) inducible GATA6 which acts as transcription factor targeting the promoter of gene stimulated during placenta, embryonic heart and gut lineage specification. The objective of this study is to investigate GATA6 expression profile in placenta cells, to determine the impact of its abnormal expression on the newborn survival. Methods: Ethical approval of CER-ISBA) was obtained prior to placenta sample collection in the hospital obstetric service. Micro-fragments of placenta tissues (n=80) were collected after delivery and lysed for GATA6 analysis with immunoblot (western blot) method. Results: We observed two isoforms of GATA6 (long L and short S isoforms). All placenta lysates of living newborn expressed the type S isoform of GATA6 (n=76). In all 80 samples there is variable expression frequency for the type L isoform of GATA6. Normal expression of Type L isoform of GATA6 was observed in 63.8% of the samples; overexpression was observed in 7.5% of the samples; low expression was in 20% of them and totally lost in 8.7% of the samples. Retrospective analysis of 6 stillborn infant charts, linked 4 of them to deficient placental GATA6. Conclusion: Our preliminary data suggested that GATA6 could be used as biomarker for embryonic and newborn survival prognostic as well as for the postnatal screening of the risk to develop congenital heart diseases and metabolic syndromes during lifespan.

Transcriptional regulation participates in heart development. However, the transcriptomes of human embryonic hearts during Carnegie stage (CS)10-CS16 have not been elucidated. Here, we found marked changes in the morphology and transcriptome of the human embryonic heart from CS10 to CS11. At CS12-CS14, the embryonic heart undergoes hypoxia-to-aerobic transformation. At CS14-CS16, transcriptome functions were related to energy metabolism, regulation of cholesterol, and processes related to inorganic substances. Moreover, the transcriptomes of cardiac progenitor cells derived from human embryonic stem cells (hESCs) most overlapped with those of human embryonic hearts at CS10. Cardiomyocytes derived from hESCs considerably overlapped with embryonic hearts at CS14-CS16. Overall, these results provide a new perspective into the characteristics of human embryonic heart development.

The adult mammalian heart is thought to be a terminally differentiated organ given the postmitotic nature of cardiomyocytes. Consequently, the potential for cardiac repair through cardiomyocyte proliferation is extremely limited. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor that is required for embryonic heart development. In this study we investigated the role of LRP6 in heart repair through regulation of cardiomyocyte proliferation. Lrp6 deficiency increased cardiomyocyte cell cycle activity in neonatal, juvenile and adult mice. Cardiomyocyte-specific deletion of Lrp6 in the mouse heart induced a robust regenerative response after myocardial infarction (MI), led to reduced MI area and improvement in left ventricular systolic function. In vivo genetic lineage tracing revealed that the newly formed cardiomyocytes in Lrp6-deficient mouse hearts after MI were mainly derived from resident cardiomyocytes. Furthermore, we found that the pro-proliferative effect of Lrp6 deficiency was mediated by the ING5/P21 signaling pathway. Gene therapy using the adeno-associated virus (AAV)9 miRNAi-Lrp6 construct promoted the repair of heart injury in mice. Lrp6 deficiency also induced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our study identifies LRP6 as a critical regulator of cardiomyocyte proliferation, which may lead to the development of a novel molecular strategy to promote myocardial regeneration and repair.

Cardiac morphogenesis requires an intricate orchestration of mechanical stress to sculpt the heart as it transitions from a straight tube to a multichambered adult heart. Mechanical properties are fundamental to this process, involved in a complex interplay with function, morphology, and mechanotransduction. In the current work, we propose a pressurization technique applied to the zebrafish atrium to quantify mechanical properties of the myocardium under passive tension. By further measuring deformation, we obtain a pressure-stretch relationship that is used to identify constitutive models of the zebrafish embryonic cardiac tissue. Two-dimensional results are compared with a three-dimensional finite element analysis based on reconstructed embryonic heart geometry. Through these steps, we found that the myocardium of zebrafish results in a stiffness on the order of 10 kPa immediately after the looping stage of development. This work enables the ability to determine how these properties change under normal and pathological heart development.