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This review will detail the embryology and morphology of the heart. We will begin with the heart's origin in the lateral plate mesoderm of the early embryo, review its fusion into a linear heart tube, followed by looping and remodeling to create a four-chambered organ with pulmonary and systemic venous inflow and pulmonary and systemic arterial outflow. We will discuss important concepts in heart development at genetic, cellular, and physiologic levels and review some key experimental models and methods important to the study of cardiac development. This review will also serve to introduce some of the congenital heart defects that can result from abnormalities in development and some of the genetic syndromes that are linked to congenital heart defects.

Purpose of Review Formation of the heart requires the coordinated addition of multiple progenitor sources which have undergone different pathways of specification and differentiation. In this review, I aim to put into context how recent studies defining cardiac progenitor heterogeneity build on our understanding of early heart development and also discuss the questions raised by this new insight. Recent Findings With the development of sequencing technologies and imaging approaches, it has been possible to define, at high temporal resolution, the molecular profile and anatomical location of cardiac progenitors at the single-cell level, during the formation of the mammalian heart. Summary Given the recent progress in our understanding of early heart development and technical advances in high-resolution time-lapse imaging and lineage analysis, we are now in a position of great potential, allowing us to resolve heart formation at previously impossible levels of detail. Understanding how this essential organ forms not only addresses questions of fundamental biological significance but also provides a blueprint for strategies to both treat and model heart disease.

Congenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects., There is a pressing need to develop representative organ-like platforms recapitulating complex in vivo phenotypes to study human development and disease in vitro. Here the authors present a method to generate human heart organoids by self-assembly using pluripotent stem cells, compare these to age-matched fetal cardiac tissues and recreate a model of pregestational diabetes.

The growth and development were Studied of the heart in common carp fishes by using several models of larvae and juvenile during artificial fertilization in the Marine Science Center hatchery of fishes. Temperature incubation at 26c°and the incubation period was 38hr.This study included the morphological and histological sides of the heart development, the primordium of the heart rudiment appeared after (22 hrs) of incubation, gradually the heart became more differentiated with the advent of ventricle and atrium in addition to the bulbus arteriosus, the histologically of the heart was appeared at age 24,28 hours from incubation as a small undifferentiated tube, while the ventricle was surrounded by two layers of cells. The atrium was surrounded with a single layer with the differentiate bulbus arteriosus, The heart at one day after hatching consist of the main parts , atrium, ventricle, sinus venous and bulbus arteriosus. Heart valves were appeared at the age of (5 – 8) days after hatching, the heart be completed once at the age of 30 days after hatching with the differentiation of the two layers of the mycardium , compact layer and trabacular layer.

Protein tyrosine phosphatase receptor-ζ1 (PTPRZ1) is a transmembrane tyrosine phosphatase receptor highly expressed in embryonic stem cells. In the present work, gene expression analyses of Ptprz1−/− and Ptprz1+/+ mice endothelial cells and hearts pointed to an unidentified role of PTPRZ1 in heart development through the regulation of heart-specific transcription factor genes. Echocardiography analysis in mice identified that both systolic and diastolic functions are affected in Ptprz1−/− compared with Ptprz1+/+ hearts, based on a dilated left ventricular (LV) cavity, decreased ejection fraction and fraction shortening, and increased angiogenesis in Ptprz1−/− hearts, with no signs of cardiac hypertrophy. A zebrafish ptprz1−/− knockout was also generated and exhibited misregulated expression of developmental cardiac markers, bradycardia, and defective heart morphogenesis characterized by enlarged ventricles and defected contractility. A selective PTPRZ1 tyrosine phosphatase inhibitor affected zebrafish heart development and function in a way like what is observed in the ptprz1−/− zebrafish. The same inhibitor had no effect in the function of the adult zebrafish heart, suggesting that PTPRZ1 is not important for the adult heart function, in line with data from the human cell atlas showing very low to negligible PTPRZ1 expression in the adult human heart. However, in line with the animal models, Ptprz1 was expressed in many different cell types in the human fetal heart, such as valvar, fibroblast-like, cardiomyocytes, and endothelial cells. Collectively, these data suggest that PTPRZ1 regulates cardiac morphogenesis in a way that subsequently affects heart function and warrant further studies for the involvement of PTPRZ1 in idiopathic congenital cardiac pathologies. NEW & NOTEWORTHY Protein tyrosine phosphatase receptor ζ1 (PTPRZ1) is expressed in fetal but not adult heart and seems to affect heart development. In both mouse and zebrafish animal models, loss of PTPRZ1 results in dilated left ventricle cavity, decreased ejection fraction, and fraction shortening, with no signs of cardiac hypertrophy. PTPRZ1 also seems to be involved in atrioventricular canal specification, outflow tract morphogenesis, and heart angiogenesis. These results suggest that PTPRZ1 plays a role in heart development and support the hypothesis that it may be involved in congenital cardiac pathologies.

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.

With the in-depth study of heart development, many human cardiomyocytes (CMs) have been generated in a laboratory environment. CMs derived from pluripotent stem cells (PSCs) have been widely used for a series of applications such as laboratory studies, drug toxicology screening, cardiac disease models, and as an unlimited resource for cell-based cardiac regeneration therapy. However, the low maturity of the induced CMs significantly impedes their applicability. Scientists have been committed to improving the maturation of CMs to achieve the purpose of heart regeneration in the past decades. In this review, we take CMs maturation as the main object of discussion, describe the characteristics of CMs maturation, summarize the key regulatory mechanism of regulating maturation and address the approaches to promote CMs maturation. The maturation of CM is gradually improving due to the incorporation of advanced technologies and is expected to continue.

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

Organoids, or miniaturized organs formed in vitro, hold potential to revolutionize how researchers approach and answer fundamental biological and pathological questions. In the context of cardiac biology, development of a bona fide cardiac organoid enables study of heart development, function, and pathogenesis in a dish, providing insight into the nature of congenital heart disease and offering the opportunity for high-throughput probing of adult heart disease and drug discovery. Recently, multiple groups have reported novel methods for generating in vitro models of the heart; however, there are substantial conceptual and methodological differences. In this review we will evaluate recent cardiac organoid studies through the lens of the core principles of organoid technology: patterned self-organization of multiple cell types resembling the in vivo organ. Based on this, we will classify systems into the following related types of tissues: developmental cardiac organoids, chamber cardiac organoids, microtissues, and engineered heart tissues. Furthermore, we highlight the interventions which allow for organoid formation, such as modulation of highly conserved cardiogenic signaling pathways mediated by developmental morphogens. We expect that consolidation and categorization of existing organoid models will help eliminate confusion in the field and facilitate progress towards creation of an ideal cardiac organoid.

Congenital Heart Disease (CHD), malformations of the heart present at birth, is the most common class of life-threatening birth defect (Hoffman (1995) [1] , Gelb (2004) [2] , Gelb (2014) [3] ). A major research challenge is to elucidate the genetic determinants of CHD and mechanistically link CHD ontogeny to a molecular understanding of heart development. Although the embryonic origins of CHD are unclear in most cases, dysregulation of cardiovascular lineage specification, patterning, proliferation, migration or differentiation have been described (Olson (2004) [4] , Olson (2006) [5], Srivastava (2006) [6] , Dunwoodie (2007) [7] , Bruneau (2008) [8]). Cardiac differentiation is the process whereby cells become progressively more dedicated in a trajectory through the cardiac lineage towards mature cardiomyocytes. Defects in cardiac differentiation have been linked to CHD, although how the complex control of cardiac differentiation prevents CHD is just beginning to be understood. The stages of cardiac differentiation are highly stereotyped and have been well-characterized (Kattman et al. (2011) [9], Wamstad et al. (2012) [10], Luna-Zurita et al. (2016) [11], Loh et al. (2016) [12], DeLaughter et al. (2016) [13]); however, the developmental and molecular mechanisms that promote or delay the transition of a cell through these stages have not been as deeply investigated. Tight temporal control of progenitor differentiation is critically important for normal organ size, spatial organization, and cellular physiology and homeostasis of all organ systems (Raff et al. (1985) [14] , Amthor et al. (1998) [15] , Kopan et al. (2014) [16] ). This review will focus on the action of signaling pathways in the control of cardiomyocyte differentiation timing. Numerous signaling pathways, including the Wnt, Fibroblast Growth Factor, Hedgehog, Bone Morphogenetic Protein, Insulin-like Growth Factor, Thyroid Hormone and Hippo pathways, have all been implicated in promoting or inhibiting transitions along the cardiac differentiation trajectory. Gaining a deeper understanding of the mechanisms controlling cardiac differentiation timing promises to yield insights into the etiology of CHD and to inform approaches to restore function to damaged hearts.

LIM-domain proteins have been shown to be associated with heart development and diseases. Systematic studies of LIM family members at the genome-wide level, which are crucial to further understand their functions in cardiac hypertrophy, are currently lacking. Here, 70 LIM genes were identified and characterised in mice. The expression patterns of LIM genes differ greatly during cardiac development and in the case of hypertrophy. Both Crip2 and Xirp2 are differentially expressed in cardiac hypertrophy and during heart failure. In addition, the hypertrophic state of cardiomyocytes is controlled by the relative expression levels of Crip2 and Xirp2. This study provides a foundation for further understanding of the special roles of LIM proteins in mammalian cardiac development and hypertrophy.

Summary During development, cells respond rapidly to intra- and intercellular signals, which induce signaling cascades regulating the activity of transcription factors at the transcriptional and/or post-translational level. The transcription factor ISL1 plays a key role in second heart field development and cardiac differentiation, and its mRNA levels are tightly regulated during cardiogenesis. Here, we show that a BMP-p38 MAPK signaling axis controls ISL1 protein function at the post-translational level. BMP-mediated activation of p38 MAPK leads to ISL1 phosphorylation at S269 by p38, which prevents ISL1 degradation and ensures its transcriptional activity during cardiogenesis. Interfering with p38 MAPK signaling leads to the degradation of ISL1 by the proteasome, resulting in defects in cardiomyocyte differentiation and impaired zebrafish and mouse heart morphogenesis and function. Given the critical role of the tight control of ISL1 activity during cardiac lineage diversification, modulation of BMP4-p38 MAPK signaling could direct differentiation into specific cardiac cell subpopulations., Highlights • ISL1 is phosphorylated by p38 MAPK at serine 269 • A BMP4-p38 MAPK signaling axis controls ISL1 protein stability • Phosphorylation of ISL1 by p38 regulates its activity during cardiogenesis • p38 Inhibition in vivo results in ISL1 degradation and second heart field defects, In this article, Jing and colleagues describe a key role of BMP4-p38 MAPK signaling in controlling ISL1 protein stability and activity during cardiogenesis through p38-mediated ISL1 phosphorylation at S269. Interfering with p38 MAPK signaling leads to the degradation of ISL1 by the proteasome, resulting in defects in ISL1+ cardiac progenitor cell differentiation and impaired heart morphogenesis and function.

AIM: The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5-12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions. METHODS AND RESULTS: We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterised these conditional knock outs using a combination of histological and molecular biology techniques.Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5-15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and post-natally. CONCLUSIONS: Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialisation and EMT/cell cycle regulation and differentiation to myogenic lineages. TRANSLATIONAL PERSPECTIVE: The molecular pathways coordinating cardiogenesis and the remodelling of the OFT are complex, and dysregulation of these pathways causes human heart defects. Our findings highlight a specific requirement for Bcar1 essential for cardiogenesis. Furthermore, the failure of OFT septation in Bcar1SM22KO mice resembles persistent truncus arteriosus (PTA), a feature of several human congenital heart diseases, including DiGeorge Syndrome. Our findings have implications for the mechanisms underlying the pathogenesis of congenital heart disease, and suggest that mice with conditional Bcar1 deletions may be useful models for dissecting mechanisms involved in the pathogenesis of human heart defects.

Aims Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD. Methods and results Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time. Conclusions In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Repairing the fish heart Although humans show minimal regenerative capability, zebrafish can regenerate their hearts through a mechanism whereby heart muscle cells (cardiomyocytes) revert to a less mature state and then proliferate to replace the damaged tissue. Ogawa et al. show that Krüppel-like factor 1 (Klf1/Eklf), a transcription factor well known for its role in red blood cell development, is an essential factor for heart regeneration in zebrafish. Klf1 is specifically expressed in cardiomyocytes after injury, and its activation is sufficient to stimulate new cardiomyocyte production without injury. This potent effect is achieved through reprogramming of gene networks regulating cardiomyocyte differentiation and mitochondrial metabolism. Science , this issue p. 201

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers (PDGFR-α, FLK1, ISL1, NKX2.5, CTNT, CX43 and MYHC-B) in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22 and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSC). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and human iPSC, with an earlier onset in the hiPSC compared to reported murine expression. Markers of immature cardiomyocytes, CTNT and MYHC-B were consistently expressed throughout E16 - E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, whilst this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm, ISL1/NKX2.5 for cardiac progenitors and CTNT/MYHC-B for immature cardiomyocytes. Further, CTNT+/ISL1+ could mark immature cardiomyocytes and CTNT+/ISL1- mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo like cardiac tissue in the future.

Increased regurgitant blood flow has been linked to endocardial cushion defects and resultant congenital heart diseases (CHDs). Prenatal alcohol exposure (PAE) has been shown to alter early blood flow resulting in abnormal endocardial cushions and CHDs. Compounds, including folic acid (FA), mitigate PAE effects and prevent CHDs, but few studies have assessed their effects on blood flow. We modeled binge drinking in quail embryos at gastrulation. Embryos were exposed to ethanol alone, FA (3.2 μg/egg) alone, and the two simultaneously. We quantified in cardiac looping stages (equivalent to 4 wk of human gestation), the regurgitant blood flow with Doppler optical coherence tomography (OCT) and endocardial cushion volumes using OCT imaging. Incidences of abnormal body curvature and heart rates were also measured. Embryos exposed to ethanol showed significantly increased regurgitant blood flow compared with controls, whereas embryos given FA with ethanol had significantly reduced regurgitant blood flow but without returning to control levels. Ethanol exposure led to significantly smaller, abnormal endocardial cushions, and the addition of FA improved their size, but they remained smaller than controls. Abnormal body curvatures after PAE were reduced in incidence but not fully prevented by FA. FA supplementation partially alleviated PAE-induced abnormal cardiovascular function and morphology. Normal blood flow and endocardial cushions are both required to produce a healthy four-chambered heart. These findings support that FA supplementation should begin early in pregnancy to prevent heart as well as neural tube defects. Investigations into the efficacy of combinations of compounds to prevent PAE-induced defects are warranted. NEW & NOTEWORTHY State-of-the-art biophotonic tools captured blood flow and endocardial cushion volumes in tiny beating quail embryo hearts, an accessible model for studying four-chambered heart development. Both hemodynamic flow and endocardial cushion volumes were altered with ethanol exposure but normalized when folic acid was introduced with ethanol. Folic acid supplementation preserved hemodynamic function that is intimately involved in sculpting the heart from the earliest stages of heart development.

Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3-7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.

During cardiogenesis, the outflow tract undergoes a complicated morphogenesis, including the re-alignment of the great blood vessels, and the separation of aorta and pulmonary trunk. The deficiency of FGF8 in the morphogenesis of outflow tract has been well studied, however, the effect of over-dosed FGF8 on the development of outflow tract remains unknown. In this study, Rosa26R-Fgf8 knock-in allele was constitutively activated by Wnt1-cre transgene in the mouse neural crest cells presumptive for the endocardial cushion of outflow tract. Surprisingly, Wnt1-cre; Rosa26R-Fgf8 mouse embryos exhibited persistent truncus arteriosus and died prior to E15.5. The cardiac neural crest cells in Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus did not degenerate as in WT controls, but proliferated into a thickened endocardial cushion and then, blocked the blood outflow from cardiac chambers into the lungs, which resulted in the embryonic lethality. Although the spiral aorticopulmonary septum failed to form, the differentiaion of the endothelium and smooth muscle in the Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus were impacted little. However, lineage tracing assay showed that the neural crest derived cells aggregated in the cushion layer, but failed to differentiate into the endothelium of Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus. Further investigation displayed the reduced p-Akt and p-Erk immunostaining, and the decreased Bmp2 and Bmp4 transcription in the endothelium of Wnt1-cre; Rosa26R-Fgf8 truncus arteriosus. Our findings suggested that Fgf8 over-expression in cardiac neural crest impaired the formation of aorticopulmonary septum by suppressing the endothelial differentiation and stimulating the proliferation of endocardial cushion cells, which implicated a novel etiology of persistent truncus arteriosus.

An essential role for cilia in the pathogenesis of congenital heart disease (CHD) has emerged from findings of a large-scale mouse forward genetic screen. High throughput screening with fetal ultrasound imaging followed by whole exome sequencing analysis recovered a preponderance of cilia related genes and cilia transduced cell signaling genes among mutations identified to cause CHD. The perturbation of left-right patterning in CHD pathogenesis is suggested by the association of CHD with heterotaxy, but also by the finding of the co-occurrence of laterality defects with CHD in birth defect registries. Many of the cilia and cilia cell signaling genes recovered were found to be related to Hedgehog signaling. Studies in mice showed cilia transduced hedgehog signaling coordinates left-right patterning with heart looping and differentiation of the heart tube. Cilia transduced Shh signaling also regulates later events in heart development, including outflow tract septation and formation of the atrioventricular septum. More recent work has shown mutations in cilia related genes may also contribute to valve disease that largely manifest in adult life. Overall, these and other findings show cilia play an important role in CHD and also in more common valve diseases.

Recent technological advancements in the field of chromatin biology have rewritten the textbook on nuclear organization. We now appreciate that the folding of chromatin in the three-dimensional space (i.e. its 3D "architecture") is non-random, hierarchical, and highly complex. While 3D chromatin structure is partially encoded in the primary sequence and thereby broadly conserved across cell types and states, a substantial portion of the genome seems to be dynamic during development or in disease. Moreover, there is growing evidence that at least some of the 3D structure of chromatin is functionally linked to gene regulation, both being modulated by and impacting on multiple nuclear processes (including DNA replication, transcription, and RNA splicing). In recent years, these new concepts have nourished several investigations about the functional role of 3D chromatin topology dynamics in the heart during development and disease. This review aims to provide a comprehensive overview of our current understanding in this field, and to discuss how this knowledge can inform further research as well as clinical practice.

Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch. Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period. Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min-1). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes. Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement.

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

Modeling Heart Development Engineering a model of the developing heart with stem cells has proved challenging. A recent study, however, provides a foundation — a single-chambered “cardioid” — for t...

How diverse cell fates and complex forms emerge and feed back to each other to sculpt functional organs remains unclear. In the developing heart, the myocardium transitions from a simple epithelium to an intricate tissue that consists of distinct layers: the outer compact and inner trabecular layers. Defects in this process, which is known as cardiac trabeculation, cause cardiomyopathies and embryonic lethality, yet how tissue symmetry is broken to specify trabecular cardiomyocytes is unknown. Here we show that local tension heterogeneity drives organ-scale patterning and cell-fate decisions during cardiac trabeculation in zebrafish. Proliferation-induced cellular crowding at the tissue scale triggers tension heterogeneity among cardiomyocytes of the compact layer and drives those with higher contractility to delaminate and seed the trabecular layer. Experimentally, increasing crowding within the compact layer cardiomyocytes augments delamination, whereas decreasing it abrogates delamination. Using genetic mosaics in trabeculation-deficient zebrafish models—that is, in the absence of critical upstream signals such as Nrg–Erbb2 or blood flow—we find that inducing actomyosin contractility rescues cardiomyocyte delamination and is sufficient to drive cardiomyocyte fate specification, as assessed by Notch reporter expression in compact layer cardiomyocytes. Furthermore, Notch signalling perturbs the actomyosin machinery in cardiomyocytes to restrict excessive delamination, thereby preserving the architecture of the myocardial wall. Thus, tissue-scale forces converge on local cellular mechanics to generate complex forms and modulate cell-fate choices, and these multiscale regulatory interactions ensure robust self-organized organ patterning. Differences in the mechanical properties of individual cardiomyocytes drive their segregation into compact versus trabecular layer, thereby transforming the myocardium in a developing heart from a simple epithelium into an intricately patterned tissue with distinct cell fates.

The most striking consequence of a heart attack is the loss of billions of heart muscle cells, alongside damage to the associated vasculature. The lost cardiovascular tissue is replaced by scar formation, which is non-functional and results in pathological remodelling of the heart and ultimately heart failure. It is, therefore, unsurprising that the heart regeneration field has centred efforts to generate new muscle and blood vessels through targeting cardiomyocyte proliferation and angiogenesis following injury. However, combined insights from embryological studies and regenerative models, alongside the adoption of -omics technology, highlight the extensive heterogeneity of cell types within the forming or re-forming heart and the significant crosstalk arising from non-muscle and non-vessel cells. In this review, we focus on the roles of fibroblasts, immune, conduction system, and nervous system cell populations during heart development and we consider the latest evidence supporting a function for these diverse lineages in contributing to regeneration following heart injury. We suggest that the emerging picture of neurologically, immunologically, and electrically coupled cell function calls for a wider-ranging combinatorial approach to heart regeneration.

The mechanisms that regulate cell-cycle arrest of cardiomyocytes during heart development are largely unknown. We have previously identified Tudor staphylococcal nuclease (Tudor-SN) as a cell-cycle regulator and have shown that its expression level was closely related to cell-proliferation capacity. Herein, we found that Tudor-SN was highly expressed in neonatal mouse myocardia, but it was lowly expressed in that of adults. Using Data Base of Transcription Start Sites (DBTSS), we revealed that Tudor-SN was a terminal oligo-pyrimidine (TOP) mRNA. We further confirmed that the translational efficiency of Tudor-SN mRNA was controlled by the mammalian target of rapamycin complex 1 (mTORC1) pathway, as revealed via inhibition of activated mTORC1 in primary neonatal mouse cardiomyocytes and activation of silenced mTORC1 in adult mouse myocardia; additionally, this result was recapitulated in H9c2 cells. We also demonstrated that the downregulation of Tudor-SN in adult myocardia was due to inactivation of the mTORC1 pathway to ensure that heart growth was in proportion to that of the rest of the body. Moreover, we revealed that Tudor-SN participated in the mTORC1-mediated regulation of cardiomyocytic proliferation, which further elucidated the correlation between Tudor-SN and the mTORC1 pathway. Taken together, our findings suggest that the translational efficiency of Tudor-SN is regulated by the mTORC1 pathway in myocardia and that Tudor-SN is involved in mTORC1-mediated regulation of cardiomyocytic proliferation and cardiac development.

Annotation. Diseases of the cardiovascular system occupy a leading place in the structure of non-infectious pathology. In the literature, small abnormalities of the development of the heart are described as abnormally located trabeculae or additional chords in the left ventricle of the heart or mitral valve prolapse. At present, the problem of cerebral hemodynamic disorders in children with them is insufficiently studied in medical sources. The aim of our study was to analyze cerebral hemodynamics using ultrasound dopplerography of cerebral vessels in children with abnormally attached chords (AAC) and mitral valve prolapse (MVP). We examined 64 children with AAC and 106 patients with MVP (among whom there were 90 with MVP I degree and 16 with MVP II degree) aged from 13 to 17 years, who formed the main group. The results were compared with the data of the control group, which included 23 almost healthy children also aged 13–17 years. All children underwent ultrasound dopplerography of extra- and intracranial vessels and veins of the brain. Evaluated the average values in the form of M±m. The values of the differences between the indicators of the two samples were evaluated by Student’s parametric criterion (t) using a special program such as Microsoft Excel. In children with MVP I deg. we found a pronounced violation of blood flow in a. vertebralis on both sides with diagnostically significant (р

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.

Valveless pumping based on Liebau mechanism entails asymmetrical positioning of the compression site relative to the attachment sites of the pump’s elastic segment to the rest of the circuit. Liebau pumping is believed to play a key role during heart development and be involved in several other physiological processes. Until now studies of Liebau pump have been limited to numerical analyses, in silico modeling, experiments using non-biological elements, and a few indirect in vivo measurements. This review aims to stimulate experimental efforts to build Liebau pumps using biologically compatible materials in order to encourage further exploration of the fundamental mechanisms behind valveless pumping and its role in organ physiology. The covered topics include the biological occurrence of Liebau pumps, the main differences between them and the peristaltic flow, and the potential uses and body sites that can benefit from implantable valveless pumps based on Liebau principle. We then provide an overview of currently available tools to build such pumps and touch upon limitations imposed by the use of biological components. We also talk about the many variables that can impact Liebau pump performance, including the concept of resonant frequencies, the shape of the flowrate-frequency relationship, the flow velocity profiles, and the Womersley numbers. Lastly, the choices of materials to build valveless impedance pumps and possible modifications to increase their flow output are briefly discussed.

Preeclampsia is a cardiovascular pregnancy complication characterised by new onset hypertension and organ damage or intrauterine growth restriction. It is one of the leading causes of maternal and fetal mortality in pregnancy globally. Short of pre-term delivery of the fetus and placenta, treatment options are limited. Consequently, preeclampsia leads to increased cardiovascular disease risk in both mothers and offspring later in life. Here we aim to examine the impact of the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia on the maternal cardiovascular system, placental and fetal heart metabolism. The surgical RUPP model was induced in pregnant rats by applying silver clips around the aorta and uterine arteries on gestational day 14, resulting in ~ 40% uterine blood flow reduction. The experiment was terminated on gestational day 19 and metabolomic profile of placentae, maternal and fetal hearts analysed using high-resolution 1H NMR spectroscopy. Impairment of uterine perfusion in RUPP rats caused placental and cardiac hypoxia and a series of metabolic adaptations: altered energetics, carbohydrate, lipid and amino acid metabolism of placentae and maternal hearts. Comparatively, the fetal metabolic phenotype was mildly affected. Nevertheless, long-term effects of these changes in both mothers and the offspring should be investigated further in the future.

Remarkable progress has been made in the direct differentiation of human pluripotent stem cells (hPSCs) into the major sources of cell types found in the heart. The understanding of cardiovascular development in vivo can provide the guidelines and help us to facilitate the process of generation of cells. In this chapter, we review the state of methods in generating cardiovascular cells from hPSCs according to our understanding of heart development. We also stress the important role of developmental biology during the differentiation from hPSCs. We then highlight the application in disease modeling, drug screening, and cell therapy based on cell transplantation. With the advances of their application, the promise of hPSCs-related products will provide new treatments for degenerative diseases of the heart in the future.

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.

Background Congenital heart disease is one of the most common cardiac malformation-related diseases worldwide. Some causative genes have been identified but can explain only a small proportion of all cases; therefore, the discovery of novel susceptibility genes and/or modifier genes for abnormal cardiac development remains a major challenge. Methods We used a single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS) was conducted to screen and quickly identify candidate genes. KIAA0196 knockout zebrafish and mice were generated by CRISPR/Cas9 to detect whether or how KIAA0196 deficiency would influence cardiac development. Results Homozygous, but not heterozygous, zebrafish and mice showed early embryonic lethality. At the embryonic stage, microscopic examination and dissection revealed pericardial edema and ventricle enlargement in homozygous zebrafish and obviously delayed cardiac development in heterozygous mice, while echocardiography and tissue staining showed that significantly decreased cardiac function, ventricle enlargement, myofibril loss, and significantly reduced trabecular muscle density were observed in adult heterozygous zebrafish and mice. Most importantly, immunostaining and electron microscopy showed that there was a significant increase in sarcomere structural disorganization and myofibril structural integrity loss in KIAA0196 mutants. Furthermore, substantial downregulation in other sarcomeric genes and proteins was detected and verified in a mouse model via transcriptome and proteomics analyses; these changes especially affected the myosin heavy or light chain (MYH or MYL) family genes. Conclusion We identified KIAA0196 for the first time as a susceptibility gene for abnormal cardiac development. KIAA0196 deficiency may cause abnormal heart development by influencing the structural integrity of myofibrils.

Heart development is one of the earliest developmental events, and its pumping action is directly linked to the intensity of development of other organs. Heart contractions mediate the circulation of the nutrients and signalling molecules to the focal points of developing embryos. In the present study, we used in vivo, ex vivo, in vitro, and in silico methods for chick embryo model to characterize and identify molecular targets under the influence of ectopic nitric oxide in reference to cardiogenesis. Spermine NONOate (SpNO) treatment of 10 μM increased the percentage of chick embryos having beating heart at 40th h of incubation by 2.2-fold (p < 0.001). In an ex vivo chick embryo culture, SpNO increased the percentage of embryos having beats by 1.56-fold (p < 0.05) compared with control after 2 h of treatment. Total body weight of SpNO-treated chick embryos at the Hamburger and Hamilton (HH) stage 29 was increased by 1.22-fold (p < 0.005). Cardiac field potential (FP) recordings of chick embryo at HH29 showed 2.5-fold (p < 0.001) increased in the amplitude, 3.2-fold (p < 0.001) increased in frequency of SpNO-treated embryos over that of the control group, whereas FP duration was unaffected. In cultured cardiac progenitors cells (CPCs), SpNO treatment decreased apoptosis and cell death by twofold (p < 0.001) and 1.7-fold (p < 0.001), respectively. Transcriptome analysis of chick embryonic heart isolated from HH15 stage pre-treated with SpNO at HH8 stage showed upregulation of genes involved in heart morphogenesis, heart contraction, cardiac cell development, calcium signalling, structure, and development whereas downregulated genes were enriched under the terms extracellular matrix, wnt pathway, and BMP pathway. The key upstream molecules predicted to be activated were p38 MAPK, MEF2C, TBX5, and GATA4 while KDM5α, DNMT3A, and HNF1α were predicted to be inhibited. This study suggests that the ectopic nitric oxide modulates the onset of cardiac development.

Endocardium is critically important for proper function of the cardiovascular system. Not only does endocardium connect the heart to blood vasculature, it also plays an important role in heart morphogenesis, valve formation, and ventricular trabeculation. The extracellular protein Fibronectin (Fn1) promotes endocardial differentiation, but the signaling pathways downstream of Fn1 that regulate endocardial development are not understood. Here, we analyzed the role of the Fibronectin receptors Integrin alpha5 (Itga5) and Integrin alpha4 (Itga4) in zebrafish heart development. We show that itga5 mRNA is expressed in both endocardium and myocardium during early stages of heart development. Through analysis of both itga5 single mutants and itga4;itga5 double mutants, we show that loss of both itga5 and itga4 results in enhanced defects in endocardial differentiation and morphogenesis compared to loss of itga5 alone. Loss of both itga5 and itga4 results in cardia bifida and severe myocardial morphology defects. Finally, we find that loss of itga5 and itga4 results in abnormally narrow anterior endodermal sheet morphology. Together, our results support a model in which Itga5 and Itga4 cooperate to promote endocardial differentiation, medial migration of endocardial and myocardial cells, and morphogenesis of anterior endoderm.

The zinc-finger protein ZBTB20 regulates development and metabolism in multiple systems, and is essential for postnatal survival in mice. However, its potential role in the cardiovascular system remains undefined. Here, we demonstrate that ZBTB20 is critically involved in the regulation of cardiac contractility and blood pressure in mice. At the age of 16 days, the relatively healthy Zbtb20-null mice exhibited hypotension without obvious change of heart rate or other evidence for heart failure. Moreover, Zbtb20 deletion led to a marked reduction in heart size, left ventricular wall thickness, and cell size of cardiomyocytes, which was largely proportional to the decreased body growth. Notably, echocardiographic and hemodynamic analyses showed that cardiac contractility was greatly impaired in the absence of ZBTB20. Mechanistically, ZBTB20 deficiency decreased cardiac ATP contents, and compromised the enzyme activity of mitochondrial complex I in heart as well as L-type calcium current density in cardiomyocytes. Furthermore, the developmental activation of some mitochondrial function-related genes was significantly attenuated in Zbtb20-null myocardium, which included Hspb8, Ckmt2, Cox7a1, Tfrc, and Ogdhl. Put together, these results suggest that ZBTB20 plays a crucial role in the regulation of heart development, energy metabolism, and contractility.

Congenital heart disease (CHD) is the most common birth defect worldwide. Long non-coding RNAs (lncRNAs) have been implicated in many diseases. However, their involvement in CHD is not well understood. This study aimed to investigate the role of dysregulated lncRNAs in CHD. We used Gene Expression Omnibus data mining, bioinformatics analysis, and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD. Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2 (HAND2). Moreover, lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation. Overall, these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5. These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

Ca2+ signaling is critical for heart development; however, the precise roles and regulatory pathways of Ca2+ transport proteins in cardiogenesis remain largely unknown. Sodium-calcium exchanger 1 (Ncx1) is responsible for Ca2+ efflux in cardiomyocytes. It is involved in cardiogenesis, while the mechanism is unclear. Here, using the forward genetic screening in zebrafish, we identified a novel mutation at a highly-conserved leucine residue in ncx1 gene (mutantLDD353/ncx1hL154P) that led to smaller hearts with reduced heart rate and weak contraction. Mechanistically, the number of ventricular but not atrial cardiomyocytes was reduced in ncx1hL154P zebrafish. These defects were mimicked by knockdown or knockout of ncx1h. Moreover, ncx1hL154P had cytosolic and mitochondrial Ca2+ overloading and Ca2+ transient suppression in cardiomyocytes. Furthermore, ncx1hL154P and ncx1h morphants downregulated cardiac transcription factors hand2 and gata4 in the cardiac regions, while overexpression of hand2 and gata4 partially rescued cardiac defects including the number of ventricular myocytes. These findings demonstrate an essential role of the novel 154th leucine residue in the maintenance of Ncx1 function in zebrafish, and reveal previous unrecognized critical roles of the 154th leucine residue and Ncx1 in the formation of ventricular cardiomyocytes by at least partially regulating the expression levels of gata4 and hand2.

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)–regulated autophagy ambra1a and ambra1b, both required for the autophagic process and during development, encode the prot...

This review summarizes current in vitro tools used for developing engineered heart muscle and studying the response of these tissues to mechanical loading changes that simulate the mechanical cues that heart muscle experiences in vivo. Advances in stem cell technology allow researchers to model genetically inherited disease phenotypes in vitro. However, one major challenge for in vitro disease modeling and drug screening is that the cardiomyocytes in simple monolayer culture are normally not exposed to the mechanical stimuli that the heart experiences in vivo. Mechanical loading is critical to heart development and pathophysiology. In vitro models have shown that cells in culture can respond to mechanical conditioning, and in some cases, this condition is critical for maturing stem cell-derived cardiomyocytes. Tools from mechanobiology have broadened the view of how physical cues guide cardiomyocyte behavior and function. This review highlights in vitro technologies for developing cardiac models and studying cardiac responses to mechanical loading.

Maturation is the last phase of heart development that prepares the organ for strong, efficient, and persistent pumping throughout the mammal’s lifespan. This process is characterized by structural, gene expression, metabolic, and functional specializations in cardiomyocytes as the heart transits from fetal to adult states. Cardiomyocyte maturation gained increased attention recently due to the maturation defects in pluripotent stem cell–derived cardiomyocyte, its antagonistic effect on myocardial regeneration, and its potential contribution to cardiac disease. Here, we review the major hallmarks of ventricular cardiomyocyte maturation and summarize key regulatory mechanisms that promote and coordinate these cellular events. With advances in the technical platforms used for cardiomyocyte maturation research, we expect significant progress in the future that will deepen our understanding of this process and lead to better maturation of pluripotent stem cell–derived cardiomyocyte and novel therapeutic strategies for heart disease.

Building a left-right (L-R) asymmetric organ requires asymmetric information. This comes from various sources, including asymmetries in embryo-scale genetic cascades (including the left-sided Nodal cascade), organ-intrinsic mechanical forces, and cell-level chirality, but the relative influence of these sources and how they collaborate to drive asymmetric morphogenesis is not understood. During zebrafish heart development, the linear heart tube extends to the left of the midline in a process known as jogging. The jogged heart then undergoes dextral (i.e. rightward) looping to correctly position the heart chambers relative to one another. Left lateralized jogging is governed by the left-sided expression of Nodal in mesoderm tissue, while looping laterality is mainly controlled by heart-intrinsic cell-level asymmetries in the actomyosin cytoskeleton. The purpose of lateralized jogging is not known. Moreover, after jogging, the heart tube returns to an almost midline position and so it is not clear whether or how jogging may impact the dextral loop. Here, we characterize a novel loss-of-function mutant in the zebrafish Nodal homolog southpaw (spaw) that appears to be a true null. We then assess the relationship between jogging and looping laterality in embryos lacking asymmetric Spaw signals. We found that the probability of a dextral loop occurring, does not depend on asymmetric Spaw signals per se, but does depend on the laterality of jogging. Thus, we conclude that the role of leftward jogging is to spatially position the heart tube in a manner that promotes robust dextral looping. When jogging laterality is abnormal, the robustness of dextral looping decreases. This establishes a cooperation between embryo-scale Nodal-dependent L-R asymmetries and organ-intrinsic cellular chirality in the control of asymmetric heart morphogenesis and shows that the transient laterality of the early heart tube has consequences for later heart morphogenetic events.

Background/Aims: Congenital heart disease (CHD) is one of the most common and severe congenital defects. The incidence of fetal cardiac malformation is increased in the context of maternal gestational diabetes mellitus (GDM). Therefore, we wanted to determine whether abnormalities in the insulin signaling pathway are associated with the occurrence of nonsyndromic CHD (ns-CHD). Methods: We used digital gene expression profiling (DGE) of right atrial myocardial tissue samples from eight ns-CHD patients and four controls. The genes potentially associated with CHD were validated by real-time fluorescence quantitative PCR analysis of right atrial myocardial tissues from 37 patients and 10 controls and the H9C2 cell line. Results: The results showed that the insulin signaling pathway, which is mediated by the SHC gene family, was inhibited in the ns-CHD patients. The expression levels of five genes (PTPRF, SHC4, MAP2K2, MKNK2, and ELK1) in the pathway were significantly down-regulated in the patients’ atrial tissues (P

The heart is lined by a single layer of mesothelial cells called the epicardium that provides important cellular contributions for embryonic heart formation. The epicardium harbors a population of progenitor cells that undergo epithelial-to-mesenchymal transition displaying characteristic conversion of planar epithelial cells into multipolar and invasive mesenchymal cells before differentiating into nonmyocyte cardiac lineages, such as vascular smooth muscle cells, pericytes, and fibroblasts. The epicardium is also a source of paracrine cues that are essential for fetal cardiac growth, coronary vessel patterning, and regenerative heart repair. Although the epicardium becomes dormant after birth, cardiac injury reactivates developmental gene programs that stimulate epithelial-to-mesenchymal transition; however, it is not clear how the epicardium contributes to disease progression or repair in the adult. In this review, we will summarize the molecular mechanisms that control epicardium-derived progenitor cell migration, and the functional contributions of the epicardium to heart formation and cardiomyopathy. Future perspectives will be presented to highlight emerging therapeutic strategies aimed at harnessing the regenerative potential of the fetal epicardium for cardiac repair.

BACKGROUND Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients. METHODS Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated. RESULTS The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients. CONCLUSIONS The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.

The phenotypic spectrum of congenital heart defects (CHDs) is contributed by both genetic and environmental factors. Their interactions are profoundly heterogeneous but may operate on common pathways as in the case of hypoxia signaling during postnatal heart development in the context of CHDs. Tetralogy of Fallot (TOF) is the most common cyanotic (hypoxemic) CHD. However, how the hypoxic environment contributes to TOF pathogenesis after birth is poorly understood. We performed Genome-wide transcriptomeanalysis on right ventricle outflow tract (RVOT) specimens from cyanotic and noncyanotic TOF. Co-expression network analysis identified gene modules specifically associated with clinical diagnosis and hypoxemia status in the TOF hearts. In particular, hypoxia-dependent induction of myocyte proliferation is associated with E2F1-mediated cell cycle regulation and repression of the WNT11-RB1 axis. Genes enriched in epithelial mesenchymal transition (EMT), fibrosis, and sarcomere were also repressed in cyanotic TOF patients. Importantly, transcription factor analysis of the hypoxia-regulated modules suggested CREB1 as a putative regulator of hypoxia/WNT11-RB1 circuit. The study providesa high-resolution landscape of transcriptome programming associated with TOF phenotypes and unveiled hypoxia-induced regulatory circuit in cyanotic TOF. Hypoxia-induced cardiomyocyte proliferation involves negative modulation of CREB1 activity upstream of the WNT11-RB1 axis. KEY MESSAGES: Genetic and environmental factors contribute to congenital heart defects (CHDs). How hypoxia contributes to Tetralogyof Fallot (TOF) pathogenesis after birth is unclear. Systems biology-based analysis revealed distinct molecular signature in CHDs. Gene expression modules specifically associated with cyanotic TOF were uncovered. Key regulatory circuits induced by hypoxia in TOF pathogenesis after birth were unveiled.

Cardiogenesis is influenced by both environmental and genetic factors, with blood flow playing a critical role in cardiac remodelling. Perturbation of any of these factors could lead to abnormal heart development and hence the formation of congenital heart defects. Although abnormal blood flow has been associated with a number of heart defects, the effects of abnormal pressure load on the developing heart gene expression profile have to date not clearly been defined. To determine the heart transcriptional response to haemodynamic alteration during development, outflow tract (OFT) banding was employed in the chick embryo at Hamburger and Hamilton stage (HH) 21. Stereological and expression studies, including the use of global expression analysis by RNA sequencing with an optimised procedure for effective globin depletion, were subsequently performed on HH29 OFT‐banded hearts and compared with sham control hearts, with further targeted expression investigations at HH35. The OFT‐banded hearts were found to have an abnormal morphology with a rounded appearance and left‐sided dilation in comparison with controls. Internal analysis showed they typically had a ventricular septal defect and reductions in the myocardial wall and trabeculae, with an increase in the lumen on the left side of the heart. There was also a significant reduction in apoptosis. The differentially expressed genes were found to be predominately involved in contraction, metabolism, apoptosis and neural development, suggesting a cardioprotective mechanism had been induced. Therefore, altered haemodynamics during development leads to left‐sided dilation and differential expression of genes that may be associated with stress and maintaining cardiac output., The pressure overloaded developing chick heart was found to have left-sided dilation and decreased apoptosis. Global gene expression profiling found changes in gene expression relating to metabolism, apoptosis, neural development and contractility, suggesting a cardioprotective response. This study provides insights into the effect that altered haemodynamics has on structure and function during cardiogenesis, increasing the understanding of the pressure overloaded developing heart.