The existence of internodal tracts (ITs) is controversial. Indeed, ITs in the cardiac conduction system (CCS), connected to the sinoatrial node (SAN), transmit electrical signals quickly to the left atrium and the atrioventricular node (AVN). Interestingly, research has suggested that the ITs and the tail of the SAN may share developmental homology. Additionally, many studies indicate that ITs blockage can lead to atrial conduction block and is associated with atrial fibrillation (AF). However, few studies have been reported on the morphogenesis, development, and function of ITs. Therefore, this paper aims to review the morphogenesis, development, and function of ITs, focusing on the regulatory mechanisms of transcription factors (TFs), such as NK2 homeobox 5 (NKX2.5), SHOX homeobox 2 (SHOX2), hyperpolarization activated cyclic nucleotide gated potassium channel 4 (HCN4), and T-box transcription factor 3 (TBX3) in the development and morphogenesis of ITs. This review also explores the causes of arrhythmias, especially atrial block, in order to provide new insights into the pathogenesis of CCS disorders.

The gene regulatory networks (GRNs) that control early heart formation are beginning to be understood, but lineage-specific GRNs remain largely undefined. We investigated networks controlled by the vital transcription factor MEF2C, with a time course of single-nucleus RNA- and ATAC-sequencing in wild-type and Mef2c -null embryos. We identified a "posteriorized" cardiac gene signature and chromatin landscape in the absence of MEF2C. Integrating our multiomics data in a deep learning-based model, we constructed developmental trajectories for each of the outflow tract, ventricular, and inflow tract segments, and alterations of these in Mef2c -null embryos. We computationally identified segment-specific MEF2C-dependent enhancers, with activity in the developing zebrafish heart. Finally, using inferred GRNs we discovered that the Mef2c -null heart malformations are partly driven by increased activity of the nuclear hormone receptor NR2F2. Our results delineate lineage-specific GRNs in the early heart tube and provide a generalizable framework for dissecting transcriptional networks governing developmental processes.

The extracellular matrix (ECM) plays a crucial role in providing structural integrity and regulating cell communication essential for organ development, homeostasis, and regeneration, including hearts. Evidence indicates that disruptions in the spatiotemporal expression or alterations in ECM components lead to cardiac malformations, including a wide range of congenital heart diseases (CHDs). Furthermore, research on injured hearts across various vertebrate species, some of which show effective regeneration while others experience irreversible fibrosis, underscores the significance of ECM molecules in cardiac regeneration. This review presents an overview of heart development and the dynamics of ECM during cardiac morphogenesis, beginning with the formation of the contractile heart tube and advancing to the development of distinct chambers separated by valves to facilitate unidirectional blood flow. Furthermore, we discuss research emphasizing the multifaceted roles of secreted molecules in mediating fibrosis and regeneration following myocardial injury.

Congenital heart defects are structural anomalies present at birth that can affect the function of the heart. Aneuploidy is a significant risk factor for congenital heart defects. Mosaic variegated aneuploidy syndrome, caused by mutations in Bub1b (encoding BubR1, a mitotic checkpoint protein), leads to congenital heart defects such as septal defects. However, the molecular rationale for how Bub1b mutations promote congenital heart defects associated with mosaic variegated aneuploidy syndrome remains unresolved.

To study morphological, structural, and cellular consequences of BubR1 deletion in the heart, we crossed mice carrying conditional alleles of Bub1b with Nkx2.5-cre mice. Single-cell RNA sequencing was carried out to determine differentially expressed genes and biological processes in various cell types present in the developing heart. Trajectory analysis was carried out to determine the differentiation trajectory of BubR1 knockout embryonic hearts. Finally, CellChat analysis provided details on the major signaling interactions that were either absent or hyperactive in the BubR1 knockout heart.

Here, we show that cardiac-specific BubR1 deletion causes embryonic lethality due to developmental stalling after cardiac looping with defects in cardiac maturation including chamber wall thickness, septation, and trabeculation. Single-cell transcriptomic profiling further revealed that the differentiation trajectory of cardiomyocytes is severely impacted with suppression of critical cardiogenesis genes. Hyperactivation of Wnt signaling in BubR1 knockout hearts indicated a disturbed homeostasis in cellular pathways essential for proper tissue morphogenesis of the heart.

Taken together, these findings reveal that BubR1 is a crucial regulator of cardiac development in vivo, which ensures the proper timing of heart morphogenesis.

Heart transplantation (HT) is the gold standard for end-stage heart disease. Donor heart preservation is an important factor that influences post-transplant success. Recently, temperature-controlled storage has demonstrated reduced primary graft dysfunction compared to standard cold storage though mechanisms are poorly understood. We hypothesized that alterations in gene expression and metabolomics offer insight into improved outcomes observed with temperature-controlled storage.

We conducted a comprehensive study to investigate the metabolic and transcriptomic responses of donor hearts preserved for 6 hours using a temperature-controlled hypothermic preservation (TCHP) system compared to conventional static cold storage (SCS). Metabolic assessments were carried out using high-resolution 1H and 31P nuclear magnetic resonance (NMR), and liquid chromatography/mass spectrometry (LC-MS) analysis on tissues obtained from various cardiac regions. Lactate, alanine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), phosphocreatine, and inorganic phosphate were measured, and metabolite ratios were calculated. Transcriptomic profiling was conducted using high throughput RNA sequencing followed by bioinformatic analysis to explore gene expression changes associated with different preservation methods.

Metabolic analyses revealed largely similar profiles between hearts preserved with TCHP and SCS. Energy metabolite ratios were comparable between preservation methods. Transcriptomic analysis unveiled a high correlation between preservation methods but also showed differential gene expression in energy metabolism and inflammation/immune-related pathways.

Our study demonstrates that TCHP maintains similar high-energy phosphate reserves to SCS but leads to alterations in gene expression of several metabolic and immunomodulatory pathways. These findings may offer important insight into reduced primary graft dysfunction observed in TCHP- hearts.

N6-methyladenosine (m6A) is the most common epigenetic modification of RNA, but whether m6A RNA methylation modulates cardiovascular development or congenital heart diseases (CHDs) has not been determined. The published high-throughput sequencing data suggested that transcripts of genes related to CHDs were prone to m6A modification, while the expression of methyltransferase-like 3 (METTL3)-involved methyltransferase complex was downregulated in mouse embryonic hearts following prenatal alcohol exposure as a critical CHD risk factor, indicating the association of insufficient m6A RNA methylation with CHDs. Using cardiovascular-specific Mettl3 knockout mice (Tagln-Cre; Mettl3flox/flox ), we observed that cardiovascular Mettl3 deficiency resulted in postnatal lethality and profound congenital cardiac defects, including left pulmonary stenosis, ventricular septal defects, and right ventricular hypoplasia. The m6A-specific methylated RNA-immunoprecipitation sequencing identified Sox4, Sox11, and Mef2a, the critical transcription factors involved in the right ventricle and outflow tract development, were the regulatory targets of METTL3-catalyzed m6A RNA methylation. Mettl3 deficiency-caused insufficient m6A RNA methylation downregulated the expression of SOX4, SOX11, and MEF2A in mouse embryonic hearts. In conclusion, cardiovascular Mettl3 deficiency directly led to congenital cardiac defects by downregulating the m6A-dependent expression of Mef2a, Sox4, and Sox11. METTL3-catalyzed m6A RNA methylation may become a potential target for preventing and treating CHDs.

While the heart works as an efficient pump, it also has a high level of adaptivity by changing its structure to maintain function during healthy and diseased states. In this Review, we present examples of structure-function relationships across species and throughout embryonic development in mammals and birds. We also summarize current research on avian models aiming at understanding how biophysical and biological mechanisms closely interact during heart formation. We conclude by underscoring similarities between cardiac adaptations and structural changes over developmental and evolutionary time scales and how understanding the mechanisms behind these adaptations can help prevent or alleviate the effects of cardiac malformations and contribute to cardiac regeneration efforts.

Cardiac development is a complex and intricate process involving numerous molecular signals and pathways. Researchers have explored cardiac development through a long journey, starting with early studies observing morphological changes and progressing to the exploration of molecular mechanisms using various molecular biology methods. Currently, advancements in stem cell technology and sequencing technology, such as the generation of human pluripotent stem cells and cardiac organoids, multi-omics sequencing, and artificial intelligence (AI) technology, have enabled researchers to understand the molecular mechanisms of cardiac development better. Many molecular signals regulate cardiac development, including various growth and transcription factors and signaling pathways, such as WNT signaling, retinoic acid signaling, and Notch signaling pathways. In addition, cilia, the extracellular matrix, epigenetic modifications, and hypoxia conditions also play important roles in cardiac development. These factors play crucial roles at one or even multiple stages of cardiac development. Recent studies have also identified roles for autophagy, metabolic transition, and macrophages in cardiac development. Deficiencies or abnormal expression of these factors can lead to various types of cardiac development abnormalities. Nowadays, congenital heart disease (CHD) management requires lifelong care, primarily involving surgical and pharmacological treatments. Advances in surgical techniques and the development of clinical genetic testing have enabled earlier diagnosis and treatment of CHD. However, these technologies still have significant limitations. The development of new technologies, such as sequencing and AI technologies, will help us better understand the molecular mechanisms of cardiac development and promote earlier prevention and treatment of CHD in the future.

The left and right ventricles of the human heart are functionally and developmentally distinct such that genetic or acquired insults can cause dysfunction in one or both ventricles resulting in heart failure. To better understand ventricle-specific molecular changes influencing heart failure development, we first performed unbiased quantitative mass spectrometry on pre-mortem non-diseased human myocardium to compare the metabolome and proteome between the normal left and right ventricles. Constituents of gluconeogenesis, glycolysis, lipogenesis, lipolysis, fatty acid catabolism, the citrate cycle and oxidative phosphorylation were down-regulated in the left ventricle, while glycogenesis, pyruvate and ketone metabolism were up-regulated. Inter-ventricular significance of these metabolic pathways was then found to be diminished within end-stage dilated cardiomyopathy and ischaemic cardiomyopathy, while heart failure-associated pathways were increased in the left ventricle relative to the right within ischaemic cardiomyopathy, such as fluid sheer-stress, increased glutamine-glutamate ratio, and down-regulation of contractile proteins, indicating a left ventricular pathological bias.

Congenital heart diseases are among the most common birth defects, significantly impacting infant health. Recent evidence suggests that exposure to endocrine-disrupting chemicals may contribute to the incidence of congenital heart diseases. This study systematically reviews and analyzes the association between maternal endocrine-disrupting chemicals exposure and congenital heart diseases.

This systematic review and meta-analysis followed the Cochrane Handbook and PRISMA guidelines. We included studies assessing the link between maternal exposure to various endocrine-disrupting chemicals and the incidence of congenital heart diseases without restricting the study design or exposure assessment methods. Data were extracted from four databases, including PubMed, Scopus, Web of Science, and Cochrane Library, up to June 2024. Quality assessment of observational studies was conducted using the Newcastle-Ottawa Scale. Statistical analysis was performed using RevMan software version 5.3, presenting results as odds ratios with 95% confidence intervals.

Fifty-nine studies were included in the meta-analysis. The pooled analysis revealed a significant association between maternal endocrine-disrupting chemical exposure and the incidence of congenital heart diseases when measured using human samples (odds ratio = 1.63, 95% confidence interval [1.35-1.97], p < 0.00001). Notably, exposure to heavy metals, polycyclic aromatic hydrocarbons, and perfluoroalkyl compounds was strongly associated with congenital heart diseases. However, non-sample-based methods showed no significant overall correlation (odds ratio = 1.08, 95% confidence interval [0.93-1.26], p = 0.30), except for housing renovation compounds, which were linked to a higher incidence of congenital heart diseases.

Maternal exposure to specific endocrine-disrupting chemicals, particularly heavy metals and polycyclic aromatic hydrocarbons, significantly increases the risk of congenital heart diseases. These findings underscore the need for preventive measures to reduce endocrine-disrupting chemicals exposure during pregnancy and further research to elucidate the underlying mechanisms.

Cell states are modulated by intrinsic driving forces such as gene expression noise and extrinsic signals from the tissue microenvironment. The distinction between intrinsic and extrinsic cell state determinants is essential for understanding the regulation of cell fate in tissues during development, homeostasis and disease. The rapidly growing availability of single-cell resolution spatial transcriptomics makes it possible to meet this challenge. However, available computational methods to infer topological tissue domains, spatially variable genes, or ligand-receptor interactions are limited in their capacity to capture cell state changes driven by crosstalk between individual cell types within the same niche. We present NiCo, a computational framework for integrating single-cell resolution spatial transcriptomics with matched single-cell RNA-sequencing reference data to infer the influence of the spatial niche on the cell state. By applying NiCo to mouse embryogenesis, adult small intestine and liver data, we demonstrate the ability to predict novel niche interactions that govern cell state variation underlying tissue development and homeostasis. In particular, NiCo predicts a feedback mechanism between Kupffer cells and neighboring stellate cells dampening stellate cell activation in the normal liver. NiCo provides a powerful tool to elucidate tissue architecture and to identify drivers of cellular states in local niches.

The vertebrate heart tube extends by progressive addition of epithelial second heart field (SHF) progenitor cells from the dorsal pericardial wall. The interplay between epithelial mechanics and genetic mechanisms during SHF deployment is unknown. Here, we present a quantitative single-cell morphometric analysis of SHF cells during heart tube extension, including force inference analysis of epithelial stress. Joint spatial Principal Component Analysis reveals that cell orientation and stress direction are the main parameters defining apical cell morphology and distinguishes cells adjacent to the arterial and venous poles. Cell shape and mechanical forces display a dynamic relationship during heart tube formation. Moreover, while the T-box transcription factor Tbx1 is necessary for cell orientation towards the arterial pole, activation of Tbx5 in the posterior SHF correlates with the establishment of epithelial stress and SHF deletion of Tbx5 relaxes the progenitor epithelium. Integrating findings from cell-scale feature patterning and mechanical stress provides new insights into cardiac morphogenesis.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are advancing cardiovascular development and disease modeling, drug testing, and regenerative therapies. However, hPSC-CM production is hindered by significant variability in the differentiation process. Establishment of early quality markers to monitor lineage progression and predict terminal differentiation outcomes would address this robustness and reproducibility roadblock in hPSC-CM production. An integrated transcriptomic and epigenomic analysis assesses how attributes of the cardiac progenitor cell (CPC) affect CM differentiation outcome. Resulting analysis identifies predictive markers of CPCs that give rise to high purity CM batches, including TTN, TRIM55, DGKI, MEF2C, MAB21L2, MYL7, LDB3, SLC7A11, and CALD1. Predictive models developed from these genes provide high accuracy in determining terminal CM purities at the CPC stage. Further, insights into mechanisms of batch failure and dominant non-CM cell types generated in failed batches are elucidated. Namely EMT, MAPK, and WNT signaling emerge as significant drivers of batch divergence, giving rise to off-target populations of fibroblasts/mural cells, skeletal myocytes, epicardial cells, and a non-CPC SLC7A11+ subpopulation. This study demonstrates how integrated multi-omic analysis of progenitor cells can identify quality attributes of that progenitor and predict differentiation outcomes, thereby improving differentiation protocols and increasing process robustness.

Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

Understanding the molecular identity of human pluripotent stem cell (hPSC)-derived cardiac progenitors and mechanisms controlling their proliferation and differentiation is valuable for developmental biology and regenerative medicine.

Here, we show that chemical modulation of histone acetyl transferases (by IQ-1) and WNT (by CHIR99021) synergistically enables the transient and reversible block of directed cardiac differentiation progression on hPSCs. The resulting stabilized cardiovascular progenitors (SCPs) are characterized by ISL1pos/KI-67pos/NKX2-5neg expression. In the presence of the chemical inhibitors, SCPs maintain a proliferation quiescent state. Upon small molecules, removal SCPs resume proliferation and concomitant NKX2-5 up-regulation triggers cell-autonomous differentiation into cardiomyocytes. Directed differentiation of SCPs into the endothelial and smooth muscle lineages confirms their full developmental potential typical of bona fide cardiovascular progenitors. Single-cell RNA-sequencing-based transcriptional profiling of our in vitro generated human SCPs notably reflects the dynamic cellular composition of E8.25-E9.25 posterior second heart field of mouse hearts, hallmarked by nuclear receptor sub-family 2 group F member 2 expression. Investigating molecular mechanisms of SCP stabilization, we found that the cell-autonomously regulated retinoic acid and BMP signalling is governing SCP transition from quiescence towards proliferation and cell-autonomous differentiation, reminiscent of a niche-like behaviour.

The chemically defined and reversible nature of our stabilization approach provides an unprecedented opportunity to dissect mechanisms of cardiovascular progenitors' specification and reveal their cellular and molecular properties.

During early embryonic development, the heart undergoes a remarkable and complex transformation, acquiring its iconic four-chamber structure whilst concomitantly contracting to maintain its essential function. The emergence of cardiac form and function involves intricate interplays between molecular, cellular, and biomechanical events, unfolding with precision in both space and time. The dynamic morphological remodelling of the developing heart renders it particularly vulnerable to congenital defects, with heart malformations being the most common type of congenital birth defect (∼35% of all congenital birth defects). This mini-review aims to give an overview of the morphogenetic processes which govern early heart formation as well as the dynamics and mechanisms of early cardiac function. Moreover, we aim to highlight some of the interplay between these two processes and discuss how recent findings and emerging techniques/models offer promising avenues for future exploration. In summary, the developing heart is an exciting model to gain fundamental insight into the dynamic relationship between form and function, which will augment our understanding of cardiac congenital defects and provide a blueprint for potential therapeutic strategies to treat disease.

Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.

The heart is the first organ formed during mammalian development and functions to distribute nutrients and oxygen to other parts of the developing embryo. Cardiomyocytes are the major cell types of the heart and provide both structural support and contractile function to the heart. The successful differentiation of cardiomyocytes during early development is under tight regulation by physical and molecular factors. We have reviewed current studies on epigenetic factors critical for cardiomyocyte differentiation, including DNA methylation, histone modifications, chromatin remodelers, and noncoding RNAs. This review also provides comprehensive details on structural and morphological changes associated with the differentiation of fetal and postnatal cardiomyocytes and highlights their differences. A holistic understanding of all aspects of cardiomyocyte development is critical for the successful in vitro differentiation of cardiomyocytes for therapeutic purposes.

Cardiomyocytes are the largest cell type that make up the heart and confer beating activity to the heart. The proper differentiation of cardiomyocytes relies on the efficient transmission and perception of differentiation cues from several signaling pathways that influence cardiomyocyte-specific gene expression programs. Signaling pathways also mediate intercellular communications to promote proper cardiomyocyte differentiation. We have reviewed the major signaling pathways involved in cardiomyocyte differentiation, including the BMP, Notch, sonic hedgehog, Hippo, and Wnt signaling pathways. Additionally, we highlight the differences between different cardiomyocyte cell lines and the use of these signaling pathways in the differentiation of cardiomyocytes from stem cells. Finally, we conclude by discussing open questions and current gaps in knowledge about the in vitro differentiation of cardiomyocytes and propose new avenues of research to fill those gaps.

Congenital heart defects (CHD) affect 1 in 100 live births and result from defects in cardiac development. Growth of the early heart tube occurs by the progressive addition of second heart field (SHF) progenitor cells to the cardiac poles. The SHF gives rise to ventricular septal, right ventricular and outflow tract myocardium at the arterial pole, and atrial, including atrial septal myocardium, at the venous pole. SHF deployment creates the template for subsequent cardiac septation and has been implicated in cardiac looping and in orchestrating outflow tract development with neural crest cells. Genetic or environmental perturbation of SHF deployment thus underlies a spectrum of common forms of CHD affecting conotruncal and septal morphogenesis. Here we review the major properties of SHF cells as well as recent insights into the developmental programs that drive normal cardiac progenitor cell addition and the origins of CHD.

Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-β, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.

The authors conducted transcardiac blood sampling in healthy subjects and subjects with heart failure with preserved ejection fraction (HFpEF) to compare cardiac metabolite and lipid substrate use. We demonstrate that fatty acids are less used by HFpEF hearts and that lipid extraction is influenced by hemodynamic factors including pulmonary pressures and cardiac index. The release of many products of protein catabolism is apparent in HFpEF compared to healthy myocardium. In subgroup analyses, differences in energy substrate use between female and male hearts were identified.

The slmap gene is alternatively spliced to generate many isoforms that are abundant in developing myocardium. The largest protein isoform SLMAP3 is ubiquitously expressed and has been linked to cardiomyopathy, Brugada syndrome and Hippo signaling. To examine any role in cardiogenesis, mice homozygous for floxed slmap allele were crossed with Nkx2.5-cre mice to nullify its expression in cardiac progenitors. Targeted deletion of the slmap gene resulted in the specific knockout (KO) of the SLMAP3 (~91 KDa) isoform without any changes in the expression of the SLMAP2 (~43 kDa) or the SLMAP1 (~35 kDa) isoforms which continued to accumulate to similar levels as seen in Wt embryonic hearts. The loss of SLMAP3 from cardiac progenitors resulted in decreased size of the developing embryonic hearts evident at E9.5 to E16.5 with four small chambers and significantly thinner left ventricles. The proliferative capacity assessed with the phosphorylation of histone 3 or with Ki67 in E12.5 hearts was not significantly altered due to SLMAP3 deficiency. The size of embryonic cardiomyocytes, marked with anti-Troponin C, revealed significantly smaller cells, but their hypertrophic response (AKT1 and MTOR1) was not significantly affected by the specific loss of SLMAP3 protein. Further, no changes in phosphorylation of MST1/2 or YAP were detected in SLMAP3-KO embryonic myocardium, ruling out any impact on Hippo signaling. Rat embryonic cardiomyocytes express the three SLMAP isoforms and their knockdown (KD) with sh-RNA, resulted in decreased proliferation and enhanced senescence but without any impact on Hippo signaling. Collectively, these data show that SLMAP is critical for normal cardiac development with potential for the various isoforms to serve compensatory roles. Our data imply novel mechanisms for SLMAP action in cardiac growth independent of Hippo signaling.

The heart and brain are the core organs of the circulation and central nervous system, respectively, and play an important role in maintaining normal physiological functions. Early neuronal and cardiac damage affects organ function. The relationship between the heart and brain is being continuously investigated. Evidence-based medicine has revealed the concept of the "heart- brain axis," which may provide new therapeutic strategies for certain diseases. Takeda protein-coupled receptor 5 (TGR5) is a metabolic regulator involved in energy homeostasis, bile acid homeostasis, and glucose and lipid metabolism. Inflammation is critical for the development and regeneration of the heart and brain during metabolic diseases. Herein, we discuss the role of TGR5 as a metabolic regulator of heart and brain development and injury to facilitate new therapeutic strategies for metabolic and ischemic diseases of the heart and brain.

The DGCR8 gene, encoding a critical miRNA processing protein, maps within the hemizygous region in patients with 22q11.2 deletion syndrome. Most patients have malformations of the cardiac outflow tract that is derived in part from the anterior second heart field (aSHF) mesoderm. To understand the function of Dgcr8 in the aSHF, we inactivated it in mice using Mef2c-AHF-Cre. Inactivation resulted in a fully penetrant persistent truncus arteriosus and a hypoplastic right ventricle leading to lethality by E14.5. To understand the molecular mechanism for this phenotype, we performed gene expression profiling of the aSHF and the cardiac outflow tract with right ventricle in conditional null versus normal mouse littermates at stage E9.5 prior to morphology changes. We identified dysregulation of mRNA gene expression, of which some are relevant to cardiogenesis. Many pri-miRNA genes were strongly increased in expression in mutant embryos along with reduced expression of mature miRNA genes. We further examined the individual, mature miRNAs that were decreased in expression along with pri-miRNAs that were accumulated that could be direct effects due to loss of Dgcr8. Among these genes, were miR-1a, miR-133a, miR-134, miR143 and miR145a, which have known functions in heart development. These early mRNA and miRNA changes may in part, explain the first steps that lead to the resulting phenotype in Dgcr8 aSHF conditional mutant embryos.

The electrical impulses that coordinate the sequential, rhythmic contractions of the atria and ventricles are initiated and tightly regulated by the specialized tissues of the cardiac conduction system. In the mature heart, these impulses are generated by the pacemaker cardiomyocytes of the sinoatrial node, propagated through the atria to the atrioventricular node where they are delayed and then rapidly propagated to the atrioventricular bundle, right and left bundle branches, and finally, the peripheral ventricular conduction system. Each of these specialized components arise by complex patterning events during embryonic development. This chapter addresses the origins and transcriptional networks and signaling pathways that drive the development and maintain the function of the cardiac conduction system.

Cardiac Myxoma is a primary tumor of heart. Its origins, rarity of the occurrence of primary cardiac tumors and how it may be related to limited cardiac regenerative potential, are not yet entirely known. This study investigates the key cardiac genes/ transcription factors (TFs) and signaling pathways to understand these important questions.

Databases including PubMed, MEDLINE, and Google Scholar were searched for published articles without any date restrictions, involving cardiac myxoma, cardiac genes/TFs/signaling pathways and their roles in cardiogenesis, proliferation, differentiation, key interactions and tumorigenesis, with focus on cardiomyocytes.

The cardiac genetic landscape is governed by a very tight control between proliferation and differentiation-related genes/TFs/pathways. Cardiac myxoma originates possibly as a consequence of dysregulations in the gene expression of differentiation regulators including Tbx5, GATA4, HAND1/2, MYOCD, HOPX, BMPs. Such dysregulations switch the expression of cardiomyocytes into progenitor-like state in cardiac myxoma development by dysregulating Isl1, Baf60 complex, Wnt, FGF, Notch, Mef2c and others. The Nkx2-5 and MSX2 contribute predominantly to both proliferation and differentiation of Cardiac Progenitor Cells (CPCs), may possibly serve roles based on the microenvironment and the direction of cell circuitry in cardiac tumorigenesis. The Nkx2-5 in cardiac myxoma may serve to limit progression of tumorigenesis as it has massive control over the proliferation of CPCs. The cardiac cell type-specific genetic programming plays governing role in controlling the tumorigenesis and regenerative potential.

The cardiomyocytes have very limited proliferative and regenerative potential. They survive for long periods of time and tightly maintain the gene expression of differentiation genes such as Tbx5, GATA4 that interact with tumor suppressors (TS) and exert TS like effect. The total effect such gene expression exerts is responsible for the rare occurrence and benign nature of primary cardiac tumors. This prevents the progression of tumorigenesis. But this also limits the regenerative and proliferative potential of cardiomyocytes. Cardiac Myxoma develops as a consequence of dysregulations in these key genes which revert the cells towards progenitor-like state, hallmark of CM. The CM development in carney complex also signifies the role of TS in cardiac cells.

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

Cardiomyocytes fail to regenerate after birth and respond to mitotic signals through cellular hypertrophy rather than cellular proliferation. Necrotic cardiomyocytes in the infarcted ventricular tissue are eventually replaced by fibroblasts, generating scar tissue. Cardiomyocyte loss causes localized systolic dysfunction. Therefore, achieving the regeneration of cardiomyocytes is of great significance for cardiac function and development. Heart development is a complex biological process. An integral cardiac developmental network plays a decisive role in the regeneration of cardiomyocytes. During this process, genetic epigenetic factors, transcription factors, signaling pathways and small RNAs are involved in regulating the developmental process of the heart. Cardiomyocyte-specific genes largely promote myocardial regeneration, among which the Nkx2.5 transcription factor is one of the earliest markers of cardiac progenitor cells, and the loss or overexpression of Nkx2.5 affects cardiac development and is a promising candidate factor. Nkx2.5 affects the development and function of the heart through its multiple functional domains. However, until now, the specific mechanism of Nkx2.5 in cardiac development and regeneration is not been fully understood. Therefore, this article will review the molecular structure, function and interaction regulation of Nkx2.5 to provide a new direction for cardiac development and the treatment of heart regeneration.

An idiopathic enlargement of the right atrium is an extremely rare cardiac malformation. There are no established guidelines for the management of this disease, especially concerning medical versus surgical therapeutic approach and the timing for an operation. We report in this case about a neonate that first was treated conservatively until the age of 5 month and finally got an operative resection of the aneurysm. After surgery, unexpected complications occurred. A second aneurysm in the left atrium was demasked. Furthermore, a progressive dilatation of both atrial chambers after resection required regular follow-up and ongoing evaluation of treatment.

T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.

Eighty percent of patients with a diagnosis of tetralogy of Fallot (TOF) do not have a known genetic etiology or syndrome. We sought to identify key molecular pathways and biological processes that are enriched in non-syndromic TOF, the most common form of cyanotic congenital heart disease, rather than single driver genes to elucidate the pathogenesis of this disease.

We undertook exome sequencing of 362 probands with non-syndromic TOF and their parents within the Pediatric Cardiac Genomics Consortium (PCGC). We identified rare (minor allele frequency <1 × 10-4), de novo variants to ascertain pathways and processes affected in this population to better understand TOF pathogenesis. Pathways and biological processes enriched in the PCGC TOF cohort were compared to 317 controls without heart defects (and their parents) from the Simons Foundation Autism Research Initiative (SFARI).

A total of 120 variants in 117 genes were identified as most likely to be deleterious, with CHD7, CLUH, UNC13C, and WASHC5 identified in two probands each. Gene ontology analyses of these variants using multiple bioinformatic tools demonstrated significant enrichment in processes including cell cycle progression, chromatin remodeling, myocyte contraction and calcium transport, and development of the ventricular septum and ventricle. There was also a significant enrichment of target genes of SOX9, which is critical in second heart field development and whose loss results in membranous ventricular septal defects related to disruption of the proximal outlet septum. None of these processes was significantly enriched in the SFARI control cohort.

Innate molecular defects in cardiac progenitor cells and genes related to their viability and contractile function appear central to non-syndromic TOF pathogenesis. Future research utilizing our results is likely to have significant implications in stratification of TOF patients and delivery of personalized clinical care.

Developmental research has attempted to untangle the exact signals that control heart growth and size, with knockout studies in mice identifying pivotal roles for Wnt and Hippo signaling during embryonic and fetal heart growth. Despite this improved understanding, no clinically relevant therapies are yet available to compensate for the loss of functional adult myocardium and the absence of mature cardiomyocyte renewal that underlies cardiomyopathies of multiple origins. It remains of great interest to understand which mechanisms are responsible for the decline in proliferation in adult hearts and to elucidate new strategies for the stimulation of cardiac regeneration. Multiple signaling pathways have been identified that regulate the proliferation of cardiomyocytes in the embryonic heart and appear to be upregulated in postnatal injured hearts. In this Review, we highlight the interaction of signaling pathways in heart development and discuss how this knowledge has been translated into current technologies for cardiomyocyte production.

Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration.

Defining the mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the proteome at critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and demonstrated a functional role for the mevalonate pathway in regulating the cell cycle of embryonic cardiomyocytes. Overall, our proteomic datasets are a resource for studying events that regulate embryonic heart development and contribute to congenital heart disease.

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.

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. 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.

During embryonic development, cell-fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors of both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study cardiopharyngeal fate specification with cellular resolution, as only two bilateral pairs of multipotent cardiopharyngeal progenitors give rise to the heart and to the pharyngeal muscles (also known as atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented and asymmetric divisions. Here, we identify the primed gene ring finger 149 related (Rnf149-r), which later becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss of Rnf149-r function impairs atrial siphon muscle morphogenesis, and downregulates Tbx1/10 and Ebf, two key determinants of pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of the loss of FGF/MAPK signaling in the cardiopharyngeal lineage, and an integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations has identified a significant overlap between candidate FGF/MAPK and Rnf149-r target genes. However, functional interaction assays suggest that Rnf149-r does not directly modulate the activity of the FGF/MAPK/Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF/MAPK signaling on shared targets, as well as on FGF/MAPK-independent targets through (a) separate pathway(s).

Domestic pigs (Sus scrofa) share many genetic, anatomical, and physiological traits with humans and therefore constitute an excellent preclinical animal model. Fundamental understanding of the cellular and molecular processes governing early porcine cardiogenesis is critical for developing advanced porcine models used for the study of heart diseases and new regenerative therapies. Here, we provide a detailed characterization of porcine cardiogenesis based on fetal porcine hearts at various developmental stages and cardiac cells derived from porcine expanded pluripotent stem cells (pEPSCs), i.e., stem cells having the potential to give rise to both embryonic and extraembryonic tissue. We notably demonstrate for the first time that pEPSCs can differentiate into cardiovascular progenitor cells (CPCs), functional cardiomyocytes (CMs), epicardial cells and epicardial-derived cells (EPDCs) in vitro. Furthermore, we present an enhanced system for whole-embryo culture which allows continuous ex utero development of porcine post-implantation embryos from the cardiac crescent stage (ED14) up to the cardiac looping (ED17) stage. These new techniques provide a versatile platform for studying porcine cardiac development and disease modeling.