Human Genetics of Atrial Septal Defect.

Although atrial septal defects (ASD) can be subdivided based on their anatomical location, an essential aspect of human genetics and genetic counseling is distinguishing between isolated and familiar cases without extracardiac features and syndromic cases with the co-occurrence of extracardiac abnormalities, such as developmental delay. Isolated or familial cases tend to show genetic alterations in genes related to important cardiac transcription factors and genes encoding for sarcomeric proteins. By contrast, the spectrum of genes with genetic alterations observed in syndromic cases is diverse. Currently, it points to different pathways and gene networks relevant to the dysregulation of cardiomyogenesis and ASD pathogenesis. Therefore, this chapter reflects the current knowledge and highlights stable associations observed in human genetics studies. It gives an overview of the different types of genetic alterations in these subtypes, including common associations based on genome-wide association studies (GWAS), and it highlights the most frequently observed syndromes associated with ASD pathogenesis.

The secretome from embryonic stem cell cardiomyogenesis: Same signals, different cellular feedbacks.

Ischemic heart diseases are a global health problem that requires the search for alternative therapies to the current treatments. Thus, an understanding of how cardiomyogenic signals can affect cellular behavior would allow us to create strategies to improve the cell recovery in damaged tissues. In this study, we aimed to evaluate the effects of the conditioned medium (CM), collected at different time points during in vitro cardiomyogenesis of human embryonic stem cells (hESCs), to direct cell behavior. We assayed different cell types to demonstrate noncytotoxic effects from the collected CM and that the CM obtained at initial time points of cardiomyogenic differentiation could promote the cell proliferation. Otherwise, the secretome derived from cardiac committed cells during cardiomyogenesis was unable to improve angiogenesis or migration in endothelial cells, and ineffective to stimulate the differentiation of cardioblasts or increase the differentiation efficiency of hESC. Therefore, we demonstrated that the effectiveness of the CM response varies depending on the cell type and the differentiation step of hESC-derived cardiomyocytes.

Targeted epigenetic modulation using a DNA-based histone deacetylase inhibitor enhances cardiomyogenesis in mouse embryonic stem cells.

The epigenome has an essential role in orchestrating transcriptional activation and modulating key developmental processes. Previously, we developed a library of pyrrole-imidazole polyamides (PIPs) conjugated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, for the purpose of sequence-specific modification of epigenetics. Based on the gene expression profile of SAHA-PIPs and screening studies using the α-myosin heavy chain promoter-driven reporter and SAHA-PIP library, we identified that SAHA-PIP G activates cardiac-related genes. Studies in mouse ES cells showed that SAHA-PIP G could enhance the generation of spontaneous beating cells, which is consistent with upregulation of several cardiac-related genes. Moreover, ChIP-seq results confirmed that the upregulation of cardiac-related genes is highly correlated with epigenetic activation, relevant to the sequence-specific binding of SAHA-PIP G. This proof-of-concept study demonstrating the applicability of SAHA-PIP not only improves our understanding of epigenetic alterations involved in cardiomyogenesis but also provides a novel chemical-based strategy for stem cell differentiation.

Hierarchically Structured Polystyrene-Based Surfaces Amplifying Fluorescence Signals: Cytocompatibility with Human Induced Pluripotent Stem Cell.

An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.

Nitro-Oleic Acid Inhibits Stemness Maintenance and Enhances Neural Differentiation of Mouse Embryonic Stem Cells via STAT3 Signaling.

Nitro-oleic acid (NO2-OA), pluripotent cell-signaling mediator, was recently described as a modulator of the signal transducer and activator of transcription 3 (STAT3) activity. In our study, we discovered new aspects of NO2-OA involvement in the regulation of stem cell pluripotency and differentiation. Murine embryonic stem cells (mESC) or mESC-derived embryoid bodies (EBs) were exposed to NO2-OA or oleic acid (OA) for selected time periods. Our results showed that NO2-OA but not OA caused the loss of pluripotency of mESC cultivated in leukemia inhibitory factor (LIF) rich medium via the decrease of pluripotency markers (NANOG, sex-determining region Y-box 1 transcription factor (SOX2), and octamer-binding transcription factor 4 (OCT4)). The effects of NO2-OA on mESC correlated with reduced phosphorylation of STAT3. Subsequent differentiation led to an increase of the ectodermal marker orthodenticle homolog 2 (Otx2). Similarly, treatment of mESC-derived EBs by NO2-OA resulted in the up-regulation of both neural markers Nestin and ß-Tubulin class III (Tubb3). Interestingly, the expression of cardiac-specific genes and beating of EBs were significantly decreased. In conclusion, NO2-OA is able to modulate pluripotency of mESC via the regulation of STAT3 phosphorylation. Further, it attenuates cardiac differentiation on the one hand, and on the other hand, it directs mESC into neural fate.

Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification.

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.

Chemical Genetics Reveals a Role of Squalene Synthase in TGFß Signaling and Cardiomyogenesis.

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

G9α-dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis-related genes in foetal mice.

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol-induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis-related genes, and histone H3K9me3 hypomethylation was observed in alcohol-treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol-induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol-induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis-related genes (MEF2C, Cx43, ANP and ß-MHC) was observed in alcohol-exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down-regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and ß-MHC overexpression. These findings suggest that G9α histone methyltransferase-mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol-induced abnormal expression cardiomyogenesis-related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.

The Distinct Function and Localization of METTL3/METTL14 and METTL16 Enzymes in Cardiomyocytes.

It has become evident that epitranscriptome events, mediated by specific enzymes, regulate gene expression and, subsequently, cell differentiation processes. We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins. The levels of METTL3 in mouse adult hearts, isolated from male and female animals, were lower in the aorta and pulmonary trunks when compared with atria, but METT14 was up-regulated in the aorta and pulmonary trunk, in comparison with ventriculi. Aging caused METTL3 down-regulation in aorta and atria in male animals. Western blot analysis in differentiated mouse embryonic stem cells (mESCs), containing 10-30 percent of cardiomyocytes, showed METTL3/METTL14 down-regulation, while the differentiation-induced increased level of METTL16 was observed in both wild type (wt) and HDAC1 depleted (dn) cells. In parallel, experimental differentiation in especially HDAC1 wild type cells was accompanied by depletion of m6A in RNA. Immunofluorescence analysis of individual cells revealed the highest density of METTL3/METTL14 in α-actinin positive cardiomyocytes when compared with the other cells in the culture undergoing differentiation. In both wt and HDAC1 dn cells, the amount of METTL16 was also up-regulated in cardiomyocytes when compared to co-cultivated cells. Together, we showed that distinct anatomical regions of the mouse adult hearts are characterized by different levels of METTL3 and METTL14 proteins, which are changed during aging. Experimental cell differentiation was also accompanied by changes in METTL-like proteins and m6A in RNA; in particular, levels and distribution patterns of METTL3/METTL14 proteins were different from the same parameters studied in the case of the METTL16 protein.

Genome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis.

The transcription factors GATA4, GATA5 and GATA6 are important regulators of heart muscle differentiation (cardiomyogenesis), which function in a partially redundant manner. We identified genes specifically regulated by individual cardiogenic GATA factors in a genome-wide transcriptomics analysis. The genes regulated by gata4 are particularly interesting because GATA4 is able to induce differentiation of beating cardiomyocytes in Xenopus and in mammalian systems. Among the specifically gata4-regulated transcripts we identified two SoxF family members, sox7 and sox18. Experimental reinstatement of gata4 restores sox7 and sox18 expression, and loss of cardiomyocyte differentiation due to gata4 knockdown is partially restored by reinstating sox7 or sox18 expression, while (as previously reported) knockdown of sox7 or sox18 interferes with heart muscle formation. In order to test for conservation in mammalian cardiomyogenesis, we confirmed in mouse embryonic stem cells (ESCs) undergoing cardiomyogenesis that knockdown of Gata4 leads to reduced Sox7 (and Sox18) expression and that Gata4 is also uniquely capable of promptly inducing Sox7 expression. Taken together, we identify an important and conserved gene regulatory axis from gata4 to the SoxF paralogs sox7 and sox18 and further to heart muscle cell differentiation.

Cardiomyogenic differentiation is fine-tuned by differential mRNA association with polysomes.

BACKGROUND: Cardiac cell fate specification occurs through progressive steps, and its gene expression regulation features are still being defined. There has been an increasing interest in understanding the coordination between transcription and post-transcriptional regulation during the differentiation processes. Here, we took advantage of the polysome profiling technique to isolate and high-throughput sequence ribosome-free and polysome-bound RNAs during cardiomyogenesis. RESULTS: We showed that polysome-bound RNAs exhibit the cardiomyogenic commitment gene expression and that mesoderm-to-cardiac progenitor stages are strongly regulated. Additionally, we compared ribosome-free and polysome-bound RNAs and found that the post-transcriptional regulation vastly contributes to cardiac phenotype determination, including RNA recruitment to and dissociation from ribosomes. Moreover, we found that protein synthesis is decreased in cardiomyocytes compared to human embryonic stem-cells (hESCs), possibly due to the down-regulation of translation-related genes. CONCLUSIONS: Our data provided a powerful tool to investigate genes potentially controlled by post-transcriptional mechanisms during the cardiac differentiation of hESC. This work could prospect fundamental tools to develop new therapy and research approaches.

Functionalization of soft materials for cardiac repair and regeneration.

Coronary artery disease is a leading cause of death in developed nations. As the disease progresses, myocardial infarction can occur leaving areas of dead tissue in the heart. To compensate, the body initiates its own repair/regenerative response in an attempt to restore function to the heart. These efforts serve as inspiration to researchers who attempt to capitalize on the natural regenerative processes to further augment repair. Thus far, researchers are exploiting these repair mechanisms in the functionalization of soft materials using a variety of growth factor-, ligand- and peptide-incorporating approaches. The goal of functionalizing soft materials is to best promote and direct the regenerative responses that are needed to restore the heart. This review summarizes the opportunities for the use of functionalized soft materials for cardiac repair and regeneration, and some of the different strategies being developed.

Sirtuin 6 mediated stem cell cardiomyogenesis on protein coated nanofibrous scaffolds.

The cellular niche provides combination of biomolecular and biophysical cues to control stem cell fate. Three-dimensional (3D) aligned nanofibrous scaffolds can effectively augment stem cell cardiomyogenesis. This work aims to understand the role of biomolecular signals from extracellular matrix (ECM) proteins and leverage them to further promote cardiomyogenesis on nanofibrous scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on 3D aligned polycaprolactone scaffolds coated with different ECM proteins. Among multiple coatings tested, collagen coated fibers were most effective in promoting cardiomyogenesis as determined from increased expression of cardiac biomarkers and intracellular calcium flux. At molecular level, enhanced differentiation on collagen coated fibers was associated with an increased level of sirtuin 6 (SIRT6). Depletion of SIRT6 using siRNA attenuated the differentiation process through activation of Wnt signaling pathway. This study, thus, demonstrates that protein coated scaffolds can augment cardiomyogenic differentiation of stem cells through a combination of topographical and biomolecular signals.

4-O-Methylhonokiol Influences Normal Cardiovascular Development in Medaka Embryo.

Although 4-O-Methylhonokiol (MH) effects on neuronal and immune cells have been established, it is still unclear whether MH can cause a change in the structure and function of the cardiovascular system. The overarching goal of this study was to evaluate the effects of MH, isolated from Magnolia grandiflora, on the development of the heart and vasculature in a Japanese medaka model in vivo to predict human health risks. We analyzed the toxicity of MH in different life-stages of medaka embryos. MH uptake into medaka embryos was quantified. The LC50 of two different exposure windows (stages 9⁻36 (0⁻6 days post fertilization (dpf)) and 25⁻36 (2⁻6 dpf)) were 5.3 ± 0.1 µM and 9.9 ± 0.2 µM. Survival, deformities, days to hatch, and larval locomotor response were quantified. Wnt 1 was overexpressed in MH-treated embryos indicating deregulation of the Wnt signaling pathway, which was associated with spinal and cardiac ventricle deformities. Overexpression of major proinflammatory mediators and biomarkers of the heart were detected. Our results indicated that the differential sensitivity of MH in the embryos was developmental stage-specific. Furthermore, this study demonstrated that certain molecules can serve as promising markers at the transcriptional and phenotypical levels, responding to absorption of MH in the developing embryo.

Quantitative Secretomics Reveals Extrinsic Signals Involved in Human Pluripotent Stem Cell Cardiomyogenesis.

Human pluripotent stem cells can be differentiated in vitro into cardiomyocytes (CMs) but the molecular mechanisms behind this process are still not fully understood. In particular, the identification of morphogens remained elusive because the mass spectrometry-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. An albumin-free cardiomyogenic differentiation medium is established in this study and applied for secretomics at seven different time points during in vitro differentiation. By this analysis 4832 proteins are identified with 1802 being significantly altered during differentiation and 431 of these are annotated as secreted. Numerous extrinsic components of Wnt, TGFß, Activin A, Nodal, BMP, or FGF signaling pathways are quantitatively assessed during differentiation. Notably, the abundance of pathway agonists is generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. It is hypothesized that TGFß, Activin A, and Nodal signaling are turned down shortly upon the initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation, and interestingly, Activin A and TGFß signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.

Cardiophysiology Illustrated by Comparing Ventricular Volumes in Healthy Adult Males and Females.

Recent advances in cardiac imaging techniques have substantially contributed to a growing interest in the analysis of global cardiac chamber dimensions and regional myocardial deformation. During the cardiac cycle, ventricular luminal volume varies due to the contraction process, which also confers a shape change including substantial alteration of long axis length, as well as rotation of the base compared to the apex. Local deformation can be assessed by strain (rate) analysis. Reviewing the present literature, it must be concluded that there is no single metric available to comprehensively characterize ventricular function. Every candidate advanced thus far has been found to incompletely reflect ventricular performance. This observation is not surprising in view of the complexity of the cardiac pump system. Additionally, sex-specific modifiers may play a role. More than three decades ago, it was shown that on average the ventricular volume is smaller in healthy women compared to matched males. Therefore, the present contribution concerns the interpretation of data derived from the healthy heart in both men and women. Starting from the classical Starling concept, we apply a simple mathematical transformation which permits an insightful representation of ventricular mechanics. Relating end-systolic volume (ESV) to end-diastolic volume creates the ventricular volume regulation graph which features the pertinent working point of an individual heart. This fundamental approach illustrates why certain proposed performance indexes cannot individually reveal the essence of ventricular systolic function. We demonstrate that particular metrics are highly interconnected and just tell us the same story in a different disguise. It is imperative to understand which associations exist and if they expectedly are (nearly) linear or frankly nonlinear. Notably, ejection fraction (EF) is primarily determined by ESV, while in turn EF is not much different from ventriculo-arterial coupling (VAC). Insight into cardiac function is promoted by identification of the paramount/essential components involved. The smaller ESV (p < 0.0001) implies that EF is higher in women and may also have consequences for VAC.

cKit+ cardiac progenitors of neural crest origin.

The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches with cKit(CreERT2/+) and Wnt1::Flpe mouse lines, we show that cKit delineates cardiac neural crest progenitors (CNC(kit)). CNC(kit) possess full cardiomyogenic capacity and contribute to all CNC derivatives, including cardiac conduction system cells. Furthermore, by modeling cardiogenesis in cKit(CreERT2)-induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNC(kit) is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit(+) cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNC(kit) contribution to myocardium.

Enhanced cardiomyogenic induction of mouse pluripotent cells by cyclic mechanical stretch.

The cardiac milieu is mechanically active with spontaneous contraction beginning from early development and persistent through maturation and homeostasis, suggesting that mechanical loading may provide a biomimetic myocardial developmental signal. In this study, we tested the role of cyclic mechanical stretch loading in the cardiomyogenesis of pluripotent murine embryonic (P19) stem cells. A Flexcell tension system was utilized to apply equiaxial stretch (12% strain, 1.25 Hz frequency) to P19 cell-derived embryoid bodies (EBs). Interestingly, while control EBs without any further stimulation did not exhibit cardiomyogenesis, stretch stimulation alone could induce P19-derived EBs to become spontaneously beating cardiomyocytes (CMs). The beating colony number, average contracting area, and beating rate, as quantified by video capturing and framed image analysis, were even increased for stretch alone case relative to those from known biochemical induction with 5-Azacytidine (5-Aza). Key CM differentiation markers, GATA4 and Troponin T, could also be detected for the stretch alone sample at comparable levels as with 5-Aza treatment. Stretch and 5-Aza co-stimulation produced in general synergistic effects in CM developments. Combined data suggest that stretch loading may serve as a potent trigger to induce functional CM development in both beating dynamics and genomic development, which is still a challenge for myocardial regenerative medicine.

Cardiomyogenesis of periodontal ligament-derived stem cells by dynamic tensile strain.

Cellular therapies for the treatment of myocardial infarction have proven to be an invaluable tool in recent years and provide encouraging evidence for the possibility to restore normal heart function. However, questions still remain as to the optimal cell source, pre-conditioning methods and delivery techniques for such an application. This study explores the use of a population of stem cells arising from the neural crest and isolated from adult human periodontal ligament along with short-term mechanical strain as an inducer of cardiomyogenesis and possibly pre-conditioning stimulus for cellular cardiomyoplasty. Cells were subjected to a short-term dynamic mechanical tension in our custom-built bioreactor and analyzed for cardiomyogenic commitment. Mechanical strain elicited a cardiomyogenic response from the cells following just 2 h of stimulation. Mechanical strain activated and translocated cardiac-specific transcription factors GATA4, MEF2C and Nkx2.5, and induced expression of the sarcomeric actin and cardiac troponin T proteins. Mechanical strain induced production of significantly higher levels of nitric oxide when compared to static controls. Elimination of elevated ROS levels by free radical scavengers completely abolished the cardiomyogenic response of the cells. MicroRNA profile changes in stretched cells were detected for 39 miRNAs with 16 of the differentially expressed miRNAs related to heart development. The use of stem cells in combination with mechanical strain prior to their delivery in vivo may pose a valuable alternative for the treatment of myocardial infarction and merits further exploration for its capacity to augment the already observed beneficial effects of cellular therapies.

Foxc1 Regulates Early Cardiomyogenesis and Functional Properties of Embryonic Stem Cell Derived Cardiomyocytes.

Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiomyocytes remains poorly understood. This is exemplified by the elusive role of the transcription factor, Foxc1, during cardiac development. The only known Foxc1 target during heart development is Tbx1. Because Foxc1 null mice contain heart mutations that are far more severe than Tbx1 null mice, it is likely that Foxc1 has additional regulatory roles during heart development. The goal of our study was to test whether Foxc1 is critical for ESC differentiation into functional cardiomyocytes through proper regulation of specific downstream gene networks. Converging evidence from Foxc1 deficient and overexpression ESC models reveals a close relationship between Foxc1 levels and early cardiomyogenic factors Isl1, Mef2c, and Nkx2.5 and also the production of functional cardiomyocytes. We show Foxc1 regulates early cardiomyogenesis during a specific window of differentiation, D4-D6. Through whole transcriptome RNA-sequencing analysis, we report pathways regulated by Foxc1 involved in cardiac function including actin cytoskeleton, cell adhesion, tight and gap junctions, and calcium signaling. Our data indicate a novel Foxc1 direct gene target, Myh7, which encodes the predominant myosin heavy chain isoform, MHCß, expressed during cardiac development. These data lead us to conclude that Foxc1 regulates both early cardiomyogenesis and the functional properties of ESC-derived cardiomyocytes. Our findings shed light on the molecular circuitry governing cardiomyogenesis that may lead to the development of better translational strategies for the use of pluripotent stem cells in regenerative medicine towards repairing damaged myocardium. Stem Cells 2016;34:1487-1500.