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Heart growth and pathological changes are accompanied by extracellular matrix‐dependent alterations in integrins and integrin‐associated proteins, suggesting their role in heart development and disease. Most of our knowledge on the involvement of integrins in heart pathology is provided by the in vivo experiments, including cardiac hypertrophy models. However, in vivo studies are limited by the complex organization of heart tissue and fail to discern cell types and particular integrins implicated in hypertrophic signalling. This problem is being addressed by isolated cardiomyocyte primary cultures, which have been successfully used in different in vitro disease models. This review aimed to analyse the general approaches to studying integrins and integrin‐associated signalling pathways in cardiac hypertrophy focusing on the in vitro systems. The lessons learned from culture experiments on the models of hypertrophy induced by stretch, stimulating factors, and/or extracellular matrix components are summarized, demonstrating the major involvement of integrin‐mediated signalling in cardiac hypertrophic response and its apparent crosstalk with signal pathways induced by stretch or hypertrophy stimulating factors. The benefits and perspectives of using cardiomyocyte primary culture as a hypertrophy model are discussed.

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.

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 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.

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.

Summary Congenital heart defects are a feature of several genetic haploinsufficiency syndromes, often involving transcriptional regulators. One property of haploinsufficient genes is their propensity for network interactions at the gene or protein level. In this article we took advantage of an online dataset of high throughput screening of mutations that are embryonic lethal in mice. Our aim was to identify new genes where the loss of function caused cardiovascular phenotypes resembling the 22q11.2 deletion syndrome models, that is, heterozygous and homozygous loss of Tbx1. One gene with a potentially haploinsufficient phenotype was identified, Setd5, thought to be involved in chromatin modification. We found murine Setd5 haploinsufficiency to be associated with double outlet right ventricle and perimembranous ventricular septal defect, although no genetic interaction with Tbx1 was detected. Conditional mutagenesis revealed that Setd5 was required in cardiopharyngeal mesoderm for progression of the heart tube through the ballooning stage to create a four‐chambered heart.

The heart is the first organ to form and function during the development of an embryo. Heart development consists of a series of events believed to be highly conserved in vertebrates. Development of heart begins with the formation of the cardiac fields followed by a linear heart tube formation. The straight heart tube then undergoes a ventral bending prior to further bending and helical torsion to form a looped heart. The looping phase is then followed by ballooning, septation, and valve formation giving rise to a four-chambered heart in avians and mammals. The looping phase plays a central role in heart development. Successful looping is essential for proper alignment of the future cardiac chambers and tracts. As aberrant looping results in various congenital heart diseases, the mechanisms of cardiac looping have been studied for several decades by various disciplines. Many groups have studied anatomy, biology, genetics, and mechanical processes during heart looping, and have proposed multiple mechanisms. Computational modeling approaches have been utilized to examine the proposed mechanisms of the looping process. Still, the exact underlying mechanism(s) controlling the looping phase remain poorly understood. Although further experimental measurements are obviously still required, the need for more integrative computational modeling approaches is also apparent in order to make sense of the vast amount of experimental data and the complexity of multiscale developmental systems. Indeed, there needs to be an iterative interaction between experimentation and modeling in order to properly find the gap in the existing data and to validate proposed hypotheses. This article is categorized under: Cardiovascular DiseasesGenetics/Genomics/Epigenetics Cardiovascular DiseasesComputational Models Cardiovascular DiseasesMolecular and Cellular Physiology.

BACKGROUND Proper development of the great vessels of the heart and septation of the cardiac outflow tract requires cardiac neural crest cells. These cells give rise to the parasympathetic cardiac ganglia, the smooth muscle layer of the great vessels, some cardiomyocytes, and the conotruncal cushions and aorticopulmonary septum of the outflow tract. Ablation of cardiac neural crest cells results in defective patterning of each of these structures. Previous studies have shown that targeted deletion of the forkhead transcription factor C2 (Foxc2), results in cardiac phenotypes similar to that derived from cardiac neural crest cell ablation. RESULTS We report that Foxc2-/- embryos on the 129s6/SvEv inbred genetic background display persistent truncus arteriosus and hypoplastic ventricles before embryonic lethality. Foxc2 loss-of-function resulted in perturbed cardiac neural crest cell migration and their reduced contribution to the outflow tract as evidenced by lineage tracing analyses together with perturbed expression of the neural crest cell markers Sox10 and Crabp1. Foxc2 loss-of-function also resulted in alterations in PlexinD1, Twist1, PECAM1, and Hand1/2 expression in association with vascular and ventricular defects. CONCLUSIONS Our data indicate Foxc2 is required for proper migration of cardiac neural crest cells, septation of the outflow tract, and development of the ventricles. Developmental Dynamics 247:1286-1296, 2018. © 2018 Wiley Periodicals, Inc.

Evidence has demonstrated that the microRNA (miR) may play a significant role in the development of congenital heart disease (CHD). Here, we explore the mechanism of microRNA-592 (miR-592) in heart development and CHD with the involvement of KCTD10 and Notch signaling pathway in a CHD mouse model. Cardiac tissues were extracted from CHD and normal mice. Immunohistochemistry staining was performed to detect positive expression rate of KCTD10. A series of inhibitor, activators, and siRNAs was introduced to verified regulatory functions for miR-592 governing KCTD10 in CHD. Furthermore, the effect of miR-592 on cell proliferation and apoptosis was also investigated. Downregulated positive rate of KCTD10 was observed in CHD mice. Downregulation of miR-592 would upregulate expression of KCTD10 and inhibit the activation of Notch signaling pathway, thus promote cell proliferation. This study demonstrates that downregulation of miR-592 prevents CHD and hypoplastic heart by inhibition of the Notch signaling pathway via negatively binding to KCTD10.

Primary cilia are small organelles projecting from the cell surface of many cell types. They play a crucial role in the regulation of various signaling pathway. In this study, we investigated the importance of cilia for heart development by conditionally deleting intraflagellar transport protein Ift88 using the col3.6-cre mouse. Analysis of col3.6;Ift88 offspring showed a wide spectrum of cardiovascular defects including double outlet right ventricle and atrioventricular septal defects. In addition, we found that in the majority of specimens the pulmonary veins did not properly connect to the developing left atrium. The abnormal connections found resemble those seen in patients with total anomalous pulmonary venous return. Analysis of mutant hearts at early stages of development revealed abnormal development of the dorsal mesocardium, a second heart field-derived structure at the venous pole intrinsically related to the development of the pulmonary veins. Data presented support a crucial role for primary cilia in outflow tract development and atrioventricular septation and their significance for the formation of the second heart field-derived tissues at the venous pole including the dorsal mesocardium. Furthermore, the results of this study indicate that proper formation of the dorsal mesocardium is critically important for the development of the pulmonary veins. Anat Rec, 302:136-145, 2019. © 2018 Wiley Periodicals, Inc.

Background Proper coronary vasculature development is essential for late-embryonic and adult heart function. The developmental regulation of coronary embryogenesis is complex and includes the coordinated activity of multiple signaling pathways. CCBE1 plays an important role during lymphangiogenesis, enhancing VEGF-C signaling, which is also required for coronary vasculature formation. However, whether CCBE1 plays a similar role during coronary vasculature development is still unknown. Here, we investigate the coronary vasculature development in Ccbe1 mutant embryos. Results We show that Ccbe1 is expressed in the epicardium, like Vegf-c, and also in the sinus venosus (SV) at the stages of its contribution to coronary vasculature formation. We also report that absence of CCBE1 in cardiac tissue inhibited coronary growth that sprouts from the SV endocardium at the dorsal cardiac wall. This disruption of coronary formation correlates with abnormal processing of VEGF-C propeptides, suggesting VEGF-C-dependent signaling alteration. Moreover, Ccbe1 loss-of-function leads to the development of defective dorsal and ventral intramyocardial vessels. We also demonstrate that Ccbe1 mutants display delayed and mispatterned coronary artery (CA) stem formation. Conclusions CCBE1 is essential for coronary vessel formation, independent of their embryonic origin, and is also necessary for peritruncal vessel growth and proper CA stem patterning. Developmental Dynamics 247:1135-1145, 2018. © 2018 Wiley Periodicals, Inc.

The ubiquitous NF-Y gene regulates the expression of different genes in various signaling pathways. However, the function of NF-Y in zebrafish heart development is largely unknown. Previously we identified a same group of cell cycle related gene cluster (CCRG) was downregulated in the embryonic hearts with impeded growth due to various stresses. The promoter regions of these CCRG genes shared a most common motif for NF-Y. Chromatin immunoprecipitation experiment demonstrated that the binding of NF-Y to its motif was real on the CCRG candidate gene promoters. Knockdown of embryonic NF-Y by morpholinos led to a small heart, mimicking the abnormal heart phenotype caused by other stresses. In parallel the expression of certain CCRG candidate genes was reduced in the NF-Y A morphant hearts exposed to malignant environments. Absence of NF-Y A also led to undermine cardiomyocyte proliferation and hence less total number of caridomyocytes per heart. Trans-AM Elisa experiment also found that in the presence of the stresses such as TCDD and TNNT2 MO, the binding capacity of NF-Y A subunit to its core motif was reduced. We conclude that NF-Y sustains proper cardiomyocyte proliferation in the heart, thus it plays a positive role in promoting early zebrafish heart growth.

The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish. Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling. Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer. Nadia Mercader was funded by the Spanish Ministry of Economy and Competitiveness through grant BFU2014-56970-P (Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016. Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigación: Proyectos I+D +i 2016, del Ministerio de Economía competitividad e Industria), and cofunding by Fondo Europeo de Desarrollo Regional (FEDER). Nadia Mercader is also supported by the European Industrial Doctorate Program EID 722427. Nadia Mercader and Julien Vermot are supported by the Swiss National Science Foundation grant ANR-SNF 310030L_182575. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 708312 (MP) and from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme: GA Nº682938. Laura Andrés-Delgado was funded (2014-16) through the postdoctoral fellowship Ayudas Postdoctorales 2013. José Luis de la Pompa was supported by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV) and RD16/0011/0021 (TERCEL) from the Spanish Ministry of Science and Innovation. The CNIC is supported by the Ministry of Science and Innovation and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí

Congenital heart disease (CHD) is the major cause of morbidity/mortality in infancy and childhood. Using a mouse model to uncover the mechanism of CHD is essential to understand its pathogenesis. However, conventional 2D phenotyping methods cannot comprehensively exhibit and accurately distinguish various 3D cardiac malformations for the complicated structure of heart cavity. Here, a new automated tool based on microcomputed tomography (micro‐CT) image data sets known as computer‐assisted cardiac cavity tracking (CACCT) is presented, which can detect the connections between cardiac cavities and identify complicated cardiac malformations in mouse hearts automatically. With CACCT, researchers, even those without expert training or diagnostic experience of CHD, can identify complicated cardiac malformations in mice conveniently and precisely, including transposition of the great arteries, double‐outlet right ventricle and atypical ventricular septal defect, whose accuracy is equivalent to senior fetal cardiologists. CACCT provides an effective approach to accurately identify heterogeneous cardiac malformations, which will facilitate the mechanistic studies into CHD and heart development., An automated tool based on microcomputed tomography (CT) images known as computer‐assisted cardiac cavity tracking (CACCT) is established to identify complicated cardiac malformations in mice. With CACCT, researchers, even those without expert training or diagnostic experience of congenital heart diseases, can identify complicated cardiac malformations effectively, including transposition of the great arteries, double‐outlet right ventricle and atypical ventricular septal defect, whose accuracy is equivalent to senior fetal cardiologists.

Background: During heart development, it has been hypothesized that apoptosis of atrioventricular canal myocardium and replacement by fibrous tissue derived from the epicardium are imperative to develop a mature atrioventricular conduction. To test this, apoptosis was blocked using an established caspase inhibitor and epicardial growth was delayed using the experimental epicardial inhibition model, both in chick embryonic hearts. Results: Chicken embryonic hearts were either treated with the peptide caspase inhibitor zVAD-fmk by intrapericardial injection in ovo (ED4) or underwent epicardial inhibition (ED2.5). Spontaneously beating embryonic hearts isolated (ED7–ED8) were then stained with voltage-sensitive dye Di-4-ANEPPS and imaged at 0.5–1 kHz. Apoptotic cells were quantified (ED5–ED7) by whole-mount LysoTracker Red and anti-active caspase 3 staining. zVAD-treated hearts showed a significantly increased proportion of immature (base to apex) activation patterns at ED8, including ventricular activation originating from the right atrioventricular junction, a pattern never observed in control hearts. zVAD-treated hearts showed decreased numbers of apoptotic cells in the atrioventricular canal myocardium at ED7. Hearts with delayed epicardial outgrowth showed also increased immature activation patterns at ED7.5 and ED8.5. However, the ventricular activation always originated from the left atrioventricular junction. Histological examination showed no changes in apoptosis rates, but a diminished presence of atrioventricular sulcus tissue compared with controls. Conclusions: Apoptosis in the atrioventricular canal myocardium and controlled replacement of this myocardium by epicardially derived HCN4-/Trop1- sulcus tissue are essential determinants of mature ventricular activation pattern. Disruption can lead to persistence of accessory atrioventricular connections, forming a morphological substrate for ventricular pre-excitation. Developmental Dynamics 247:1033-1042, 2018. © 2018 Wiley Periodicals, Inc.

Background During the final stages of heart development the myocardium grows and becomes vascularized by means of paracrine factors and cell progenitors derived from the epicardium. There is evidence to suggest that retinoic acid (RA), a metabolite of vitamin A, plays an important role in epicardial-based developmental programming. However, the consequences of altered RA-signaling in coronary development have not been systematically investigated. Results We explored the developmental consequences of altered RA-signaling in late cardiogenic events that involve the epicardium. For this, we used a model of embryonic RA excess based on mouse embryos deficient in the retinaldehyde reductase DHRS3, and a complementary model of embryonic RA deficiency based on pharmacological inhibition of RA synthesis. We found that alterations in embryonic RA signaling led to a thin myocardium and aberrant coronary vessel formation and remodeling. Both excess, and deficient RA-signaling are associated with reductions in ventricular coverage and density of coronary vessels, altered vessel morphology, and impaired recruitment of epicardial-derived mural cells. Using a combined transcriptome and proteome profiling approach, we found that RA treatment of epicardial cells influenced key signaling pathways relevant for cardiac development. Conclusions Epicardial RA-signaling plays critical roles in the development of the coronary vasculature needed to support myocardial growth. Developmental Dynamics 247:976-991, 2018. © 2018 Wiley Periodicals, Inc.

Myocardial infarction is a primary contributor towards the global burden of cardiovascular disease. Rather than repairing the existing damage of myocardial infarction, current treatments only address the symptoms of the disease and reducing the risk of a secondary infarction. Cardiac regenerative capacity is dependent on cardiomyocyte proliferation, which concludes soon after birth in humans and precocial species such as sheep. Human fetal cardiac tissue has some ability to repair following tissue damage, whereas a fully matured human heart has minimal capacity for cellular regeneration. This is in contrast to neonatal mice and adult zebrafish hearts, which retain the ability to undergo cardiomyocyte proliferation and can regenerate cardiac tissue after birth. In mice and zebrafish models, microRNAs (miRNAs) have been implicated in the regulation of genes involved in cardiac cell cycle progression and regeneration. However, the significance of miRNA regulation in cardiomyocyte proliferation for humans and other large mammals, where the timing of heart development in relation to birth is similar, remains unclear. miRNAs may be valuable targets for therapies that promote cardiac repair after injury. Therefore, elucidating the role of specific miRNAs in large animals, where heart development closely resembles that of humans, remains vitally important for identifying therapeutic targets that may be translated into clinical practice focused on tissue repair.

The NR2F2 gene plays an important role in angiogenesis and heart development. Moreover, this gene is involved in organogenesis in many other organs in mouse models. Variants in this gene have been reported in a number of patients with nonsyndromic atrioventricular septal defect, and in one patient with congenital heart defect and dysmorphic features. Here we report an 11-month-old Caucasian male with global developmental delay, dysmorphic features, coarctation of the aorta, and ventricular septal defect. He was later found to have a pathogenic mutation in the NR2F2 gene by whole exome sequencing. This is the second instance in which an NR2F2 mutation has been identified in a child with a congenital heart defect and other anomalies. This case suggests that some variants in NR2F2 may cause syndromic forms of congenital heart defect.

Tree swallow (Tachycineta bicolor) eggs from 2 uncontaminated sites, the Patuxent Research Refuge (Laurel, MD, USA) and the Cobleskill Reservoir (Cobleskill, NY, USA) were dosed with polychlorinated biphenyl (PCB) 77 to evaluate effects on the developing cardiovascular system. To ensure embryonic viability, treatments were administered into the air cell at embryonic day 2.5 including: untreated (control), vehicle (filtered sterilized fatty acid mixture), 100 ng/g and 1000 ng/g egg. Eggs were dosed in the field with 0.2 μL/egg, returned to the nest, collected at embryonic day 13, hatched in the laboratory, and necropsied. The PCB 77-treated hatchlings were compared with uninjected, vehicle-injected, and environmentally exposed hatchlings collected from a PCB-contaminated Upper Hudson River (NY, USA) site. The PCB 77-treated embryos showed no effects on hatching success or hatchling mortality, heart index, or morphological measures of 4 distinct heart layers (heart width, length, septal thickness, total and ventricular cavity area) compared with controls. Hatchlings that had received PCB 77 exhibited increased incidence of a cardiomyopathy and absence of the ventricular heart wall compact layer (Chi square test; p

BACKGROUND In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes. RESULTS We demonstrate that loss of hace1 in zebrafish via morpholino knockdown results in cardiac deformities, specifically a looping defect, where the heart is either tubular or "inverted". Whole-mount in situ hybridization of cardiac markers shows distinct abnormalities in ventricular morphology and atrioventricular valve formation in the hearts of these morphants, as well as increased expression of rac1. Importantly, this phenotype appears to be directly related to Nox enzyme-dependent ROS production, as both genetic inhibition by nox1 and nox2 morpholinos or pharmacologic rescue using ROS scavenging agents restores normal cardiac structure. CONCLUSIONS Our study demonstrates that HACE1 is critical in the normal development and proper function of the vertebrate heart via a ROS-dependent mechanism. Developmental Dynamics 247:289-303, 2018. © 2017 Wiley Periodicals, Inc.

Background: The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the non-catalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. Results: Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. Conclusion: Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. This article is protected by copyright. All rights reserved.

Signal transduction through multiple distinct pathways regulates and orchestrates the numerous biological processes comprising heart development. This review outlines the roles of the FGFR, EGFR, Wnt, BMP, Notch, Hedgehog, Slit/Robo, and other signaling pathways during four sequential phases of Drosophila cardiogenesis-mesoderm migration, cardiac mesoderm establishment, differentiation of the cardiac mesoderm into distinct cardiac cell types, and morphogenesis of the heart and its lumen based on the proper positioning and cell shape changes of these differentiated cardiac cells-and illustrates how these same cardiogenic roles are conserved in vertebrates. Mechanisms bringing about the regulation and combinatorial integration of these diverse signaling pathways in Drosophila are also described. This synopsis of our present state of knowledge of conserved signaling pathways in Drosophila cardiogenesis and the means by which it was acquired should facilitate our understanding of and investigations into related processes in vertebrates. Developmental Dynamics 246:641-656, 2017. © 2017 Wiley Periodicals, Inc.

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube.

Background - Understanding how molecular and physical cues orchestrate vascular morphogenesis is a challenge for developmental biology. Only little attention has been paid to the impact of mechanical stress caused by tissue growth on early blood distribution. Here we study the peripheral accumulation of blood in the chicken embryonic yolk sac, which precedes sinus vein formation. Results - We report that blood accumulation starts prior to heart-induced blood circulation. We hypothesized that the driving force for the primitive blood flow is a growth-induced gradient of tissue pressure in the yolk sac mesoderm. Therefore, we studied embryos in which heart development was arrested after two days of incubation, and found that yolk sac growth and blood peripheral accumulation still occurred. This suggests that tissue growth is sufficient to initiate the flow and the formation of the sinus vein, whereas heart contractions are not required. We designed a simple mathematical model which makes explicit the growth-induced pressure gradient and the subsequent blood accumulation, and show that growth can indeed account for the observed blood accumulation. Conclusions - This study shows that tissue growth pressure can drive early blood flow, and suggests that the mechanical environment, beyond hemodynamics, can contribute to vascular morphogenesis. This article is protected by copyright. All rights reserved.

The Zebrafish has emerged to become a powerful vertebrate animal model for cardiovascular research in recent years. Its advantages include easy genetic manipulation, transparency, small size, low cost, and the ability to survive without active circulation at early stages of development. Sequencing the whole genome and identifying ortholog genes with human genome made it possible to induce clinically relevant cardiovascular defects via genetic approaches. Heart function and disturbed hemodynamics need to be assessed in a reliable manner for these disease models in order to reveal the mechanobiology of induced defects. This effort requires precise determination of blood flow patterns as well as hemodynamic stress (i.e., wall shear stress and pressure) levels within the developing heart. While traditional approach involves time-lapse brightfield microscopy to track cell and tissue movements, in more recent studies fast light-sheet fluorescent microscopes are utilized for that purpose. Integration of more complicated techniques like particle image velocimetry and computational fluid dynamics modeling for hemodynamic analysis holds a great promise to the advancement of the Zebrafish studies. Here, we discuss the latest developments in heart function and hemodynamic analysis for Zebrafish embryos and conclude with our future perspective on dynamic analysis of the Zebrafish cardiovascular system. Developmental Dynamics 246:868-880, 2017. © 2017 Wiley Periodicals, Inc.

Oral-facial-digital syndrome type 1 (OFD1; OMIM# 311200) is an X-linked dominant ciliopathy caused by mutations in the OFD1 gene. This condition is characterized by facial anomalies and abnormalities of oral tissues, digits, brain, and kidneys. Almost all affected patients are female, as OFD1 is presumed to be lethal in males, mostly in the first or second trimester of pregnancy. Live born males with OFD1 are a rare occurrence, with only five reported patients to date. In four patients the presence of a congenital heart defect (CHD) was observed. Here, we report an affected male fetus with a hemizygous de novo mutation in OFD1 (c.2101C>T; p.(Gln701*)). Ultrasound examination demonstrated severe hydrocephalus, a hypoplastic cerebellum and a hypoplastic left ventricle of the heart. The pregnancy was terminated at 16 weeks of gestation because of poor prognosis. Post-mortem examination of the fetus confirmed severe hypoplasia of the left ventricle of the heart. We emphasize that CHDs should be included in the phenotypic spectrum of OFD1 in males. This justifies molecular analysis of OFD1 when CHD is encountered prenatally in combination with one or more phenotypic features previously described in the OFD1 gene alteration spectrum. The underlying pathogenesis of CHD in OFD1 (and other ciliopathies) probably involves dysfunction of the primary cilia regarding coordination of left-right signalling during early heart development. Whether these CHDs wholly or partly result from defective left right signalling, in which different types of cilia are known to play a critical role, remains a topic of research.

Background Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. Methods We genotyped 32 HRHS cases identified from all New York State live births (1998–2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20 Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3, and Childrens Hospital of Philadelphia database. Results We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16-2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1−/− mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5 Mb deletion associated with Williams-Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24 Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. Conclusion To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. Birth Defects Research 109:16–26, 2017. © 2016 Wiley Periodicals, Inc.

Scientists have studied organs and their development for centuries and, along that path, described models and mechanisms explaining the developmental principles of organogenesis. In particular, with respect to the heart, new fundamental discoveries are reported continuously that keep changing the way we think about early cardiac development. These discoveries are driven by the need to answer long-standing questions regarding the origin of the earliest cells specified to the cardiac lineage, the differentiation potential of distinct cardiac progenitor cells, and, very importantly, the molecular mechanisms underlying these specification events. As evidenced by numerous examples, the wealth of developmental knowledge collected over the years has had an invaluable impact on establishing efficient strategies to generate cardiovascular cell types ex vivo, from either pluripotent stem cells or via direct reprogramming approaches. The ability to generate functional cardiovascular cells in an efficient and reliable manner will contribute to therapeutic strategies aimed at alleviating the increasing burden of cardiovascular disease and morbidity. Here we will discuss the recent discoveries in the field of cardiac progenitor biology and their translation to the pluripotent stem cell model to illustrate how developmental concepts have instructed regenerative model systems in the past and promise to do so in the future. Developmental Dynamics 245:1130-1144, 2016. © 2016 Wiley Periodicals, Inc.

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.—Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Background: 14-3-3e plays an important role in the maturation of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via p27kip1. However, additional cardiac defects are possible given the ubiquitous expression pattern of this protein. Results: Germ line deletion of 14-3-3e led to malalignment of both the outflow tract (OFT) and atrioventricular (AV) cushions, with resulting tricuspid stenosis and atresia, mitral valve abnormalities, and perimembranous ventricular septal defects (VSDs). We confirmed myocardial non-compaction and detected a spongy septum with muscular VSDs and blebbing of the epicardium. These defects were associated with abnormal patterning of p27kip1 expression in the subendocardial and possibly the epicardial cell populations. In addition to abnormal pharyngeal arch artery patterning, we found deep endocardial recesses and paucity of intramyocardial coronary vasculature as a result of defective coronary plexus remodeling. Conclusions: The malalignment of both endocardial cushions provides a new explanation for tricuspid and mitral valve defects, while myocardial non-compaction provides the basis for the abnormal coronary vasculature patterning. These abnormalities might arise from p27kip1 dysregulation and a resulting defect in epithelial-to-mesenchymal transformation. These data suggest that 14-3-3e, in addition to left ventricular non-compaction (LVNC), might be linked to different forms of congenital heart disease (CHD). Developmental Dynamics 245:1107–1123, 2016. © 2016 Wiley Periodicals, Inc.

D-transposition of the great arteries (TGA) is one of the most common conotruncal heart defects at birth and is characterized by a discordant ventriculoarterial connection with a concordant atrioventricular connection. The morphological etiology of TGA is an inverted or arrested rotation of the heart outflow tract (OFT, conotruncus), by which the aorta is transposed in the right ventral direction to the pulmonary trunk. The rotational defect of the OFT is thought to be attributed to hypoplasia of the subpulmonic conus, which originates from the left anterior heart field (AHF) residing in the mesodermal core of the first and second pharyngeal arches. AHF, especially on the left, at the early looped heart stage (corresponding to Carnegie stage 10-11 in the human embryo) is one of the regions responsible for the impediment that causes TGA morphology. In human or experimentally produced right isomerism, malposition of the great arteries including D-TGA is frequently associated. Mutations in genes involving left-right (L-R) asymmetry, such as NODAL, ACTRIIB and downstream target FOXH1, have been found in patients with right isomerism as well as in isolated TGA. The downstream pathways of Nodal-Foxh1 play a critical role not only in L-R determination in the lateral plate mesoderm but also in myocardial specification and differentiation in the AHF, suggesting that TGA is a phenotype in heterotaxia as well as the primary developmental defect of the AHF.

The heart is the first organ to form and undergoes adaptive remodelling with age. Ventricular hypertrophy is one such adaptation, which allows the heart to cope with an increase in cardiac demand. This adaptation is necessary as part of natural growth from foetal life to adulthood. It may also occur in response to resistance in blood flow due to various insults on the heart and vessels that accumulate with age. The heart can only compensate to this increase in workload to a certain extent without losing its functional architecture, ultimately resulting in heart failure. Many genes have been implicated in cardiac hypertrophy, however none have been shown conclusively to be responsible for pathological cardiac hypertrophy. MicroRNAs offer an alternative mechanism for cellular regulation by altering gene expression. Since 1993 when the function of a non-coding DNA sequence was first discovered in the model organism Caenorhabditis elegans, many microRNAs have been implicated in having a central role in numerous physiological and pathological cellular processes. The level of control these antisense oligonucleotides offer can often be exploited to manipulate the expression of target genes. Moreover, altered levels of microRNAs can serve as diagnostic biomarkers, with the prospect of diagnosing a disease process as early as during foetal life. Therefore, it is vital to ascertain and investigate the function of microRNAs that are involved in heart development and subsequent ventricular remodelling. Here we present an overview of the complicated network of microRNAs and their target genes that have previously been implicated in cardiogenesis and hypertrophy. It is interesting to note that microRNAs in both of these growth processes can be of possible remedial value to counter a similar disease pathophysiology.

Aim We analysed the effects of acute temperature change on the beating rate, conduction properties and calcium transients in the chick embryonic heart in vitro and in ovo. Methods The effects of temperature change (34, 37 and 40 °C) on calcium dynamics in isolated ED4 chick hearts in vitro were investigated by high-speed calcium optical imaging. For comparison and validation of in vitro measurements, experiments were also performed in ovo using videomicroscopy. Artificial stimulation experiments were performed in vitro and in ovo to uncover conduction limits of heart segments. Results Decrease in temperature from 37 to 34 °C in vitro led to a 22% drop in heart rate and unchanged amplitude of Ca2+ transients, compared to a 25% heart rate decrease in ovo. Increase in temperature from 37 to 40 °C in vitro and in ovo led to 20 and 23% increases in heart rate, respectively, and a significant decrease in amplitude of Ca2+ transients (atrium −35%, ventricle −38%). We observed a wide spectrum of arrhythmias in vitro, of which the most common was atrioventricular (AV) block (57%). There was variability of AV block locations. Pacing experiments in vitro and in ovo suggested that the AV blocks were likely caused by relative tissue hypoxia and not by the tachycardia itself. Conclusion The pacemaker and AV canal are the most temperature-sensitive segments of the embryonic heart. We suggest that the critical point for conduction is the connection of the ventricular trabecular network to the AV canal.

Heart disease is the number one cause of morbidity and mortality in the world and is a major health and economic burden, costing the United States Health Care System more than $200 billion annually. A major cause of heart disease is the massive loss or dysfunction of cardiomyocytes caused by myocardial infarctions and hypertension. Due to the limited regenerative capacity of the heart, much research has focused on better understanding the process of differentiation toward cardiomyocytes. This review will highlight what is currently known about cardiac cell specification during mammalian development, areas of controversy, cellular sources of cardiomyocytes, and current and potential uses of stem cell derived cardiomyocytes for cardiac therapies. Developmental Dynamics 245:751-761, 2016. © 2016 Wiley Periodicals, Inc.

Background Coronary artery (CA) stems connect the ventricular coronary tree with the aorta. Defects in proximal CA patterning are a cause of sudden cardiac death. In mice lacking Tbx1, common arterial trunk is associated with an abnormal trajectory of the proximal left CA. Here we investigate CA stem development in wild-type and Tbx1 null embryos. Results Genetic lineage tracing reveals that limited outgrowth of aortic endothelium contributes to proximal CA stems. Immunohistochemistry and fluorescent tracer injections identify a periarterial vascular plexus present at the onset of CA stem development. Transplantation experiments in avian embryos indicate that the periarterial plexus originates in mesenchyme distal to the outflow tract. Tbx1 is required for the patterning but not timing of CA stem development and a Tbx1 reporter allele is expressed in myocardium adjacent to the left but not right CA stem. This expression domain is maintained in Sema3c(-/-) hearts with a common arterial trunk and leftward positioned CA. Ectopic myocardial differentiation is observed on the left side of the Tbx1(-/-) common arterial trunk. Conclusions A periarterial plexus bridges limited outgrowth of the aortic endothelium with the ventricular plexus during CA stem development. Molecular differences associated with left and right CA stems provide new insights into the etiology of CA patterning defects.

Wnt signaling plays an essential role in development and differentiation. Heart development is initiated with the induction of precardiac mesoderm requiring the tightly and spatially controlled regulation of canonical and noncanonical Wnt signaling pathways. The role of Wnt signaling in subsequent development of the heart fields is to a large extent unclear. We will discuss the role of Wnt signaling in the development of the arterial and venous pole of the heart, highlighting the dual roles of Wnt signaling with respect to its time- and dosage-dependent effects and the balance between the canonical and noncanonical signaling. Canonical signaling appears to be involved in retaining the cardiac precursors in a proliferative and precursor state, whereas noncanonical signaling promotes their differentiation. Thereafter, both canonical and noncanonical signaling regulate specific steps in differentiation of the cardiac compartments. Because heart development is a contiguous, rather than a sequential, process, analyses tend only to show a single timeframe of development. The repetitive alternating and reciprocal effect of canonical and noncanonical signaling is lost when studied in homogenates. Without the simultaneous in vivo visualization of the different Wnt signaling pathways, the mechanism of Wnt signaling in heart development remains elusive.

Although considerable advances in our understanding of mammalian and avian embryonic coronary development have occurred during the last decade, our current knowledge of this topic in humans is limited. Accordingly, the aim of this study was to determine if the development of the human coronary vasculature in humans is like that of other mammals and avians. The data document a progression of events involving mesenchymal cell-containing villi from the proepicardium, establishment of blood islands and a capillary network. The major finding of the study is direct evidence that the capillary plexus associated with spindle cells and erythroblasts invades the base of the aorta to form coronary ostia. A role for the dorsal mesocardium is also indicated by the finding that cells from this region are continuous with the aorta and pulmonary artery. The development of the tunica media of the coronary arteries follows the same base-apex progression as in other species, with the development of branches occurring late in the embryonic period. The fetal period is characterized by 1) growth and a numerical increase in the smallest arterial branches, veins, and venules, 2) innervation of arteries, and 3) inclusion of elastic fibers in the tunica media of the coronary arteries and development of the tunica adventitia. In conclusion, the data demonstrate that the development of the coronary system in humans is similar to that of other mammalian and avian species, and for the first time documents that the formation of the ostia and coronary stems in humans occurs by ingrowth of a vascular plexus and associated cells from the epicardium.

Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6-hydroxydopamine (6-OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β-adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β-adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.

Background: Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations affecting the outflow tract (OFT). To address the cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. Results: By embryonic day 13.5, all rescued Raldh2(-/-) hearts exhibit severe, reproducible OFT septation defects, although wild-type and Raldh2(+/-) littermates have normal hearts. Cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2(-/-) mutant embryos but ectopically located in the periphery of the endocardial cushions, rather than immediately underlying the endocardium. Excess mesenchyme was generated by Raldh2(-/-) mutant endocardium, which displaced cNCC derivatives from their subendocardial, medial position. Conclusions: RA signaling affects not only cNCC numbers but also their position relative to endocardial mesenchyme during the septation process. Our study shows that inappropriate coordination between the different cell types of the OFT perturbs its morphogenesis and leads to a severe congenital heart defect, persistent truncus arteriosus. Developmental Dynamics 245:388-401, 2016. (c) 2015 Wiley Periodicals, Inc.

Knowledge of cardiac development can provide the basis for understanding the morphogenesis of congenital cardiac malformations. Only recently, however, has the quality of information regarding cardiac embryology been sufficient to justify this approach. In this review, we show how such knowledge of development of the normal atrial and ventricular septal structures underscores the interpretation of the lesions that provide the basis for interatrial and interventricular shunting of blood. We show that current concepts of atrial septation, which frequently depend on a suggested formation of an extensive secondary septum, are simplistic. There are additional contributions beyond growth of the primary septum, but the new tissue is added to form the ventral buttress of the definitive atrial septum, rather than its cranial margin, as is usually depicted. We show that the ventricular septum possesses muscular and membranous components, with the entirety of the muscular septum produced concomitant with the so-called ballooning of the apical ventricular component. It is expansion of the atrioventricular canal that creates the inlet of the right ventricle, with no separate formation of an "inlet" septum. The proximal parts of the outflow cushions initially form a septal structure between the developing ventricular outlets, but this becomes converted into the free-standing muscular subpulmonary infundibulum as the aortic outlet is transferred to the left ventricle. These features of normal development are then shown to provide the basis for understanding of the channels that provide the means for interatrial and interventricular shunting.