Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.

The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.

A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.

Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood owing, in part, to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single-cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently expressed mesodermal transcription factor, is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mislocalized, prompting us to investigate the scope of the role of Mesp1 in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and crucial cardiac transcription factors, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single-cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis.

Dgcr8 functions in the secondary heart field for outflow tract and right ventricle development in mammals.

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.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

Trisomy 21-driven metabolite alterations are linked to cellular injuries in Down syndrome.

Down syndrome (DS) arises from a genetic anomaly characterized by an extra copy of chromosome 21 (exCh21). Despite high incidence of congenital diseases among DS patients, direct impacts of exCh21 remain elusive. Here, we established a robust DS model harnessing human-induced pluripotent stem cells (hiPSCs) from mosaic DS patient. These hiPSC lines encompassed both those with standard karyotype and those carrying an extra copy of exCh21, allowing to generate isogenic cell lines with a consistent genetic background. We unraveled that exCh21 inflicted disruption upon the cellular transcriptome, ushering in alterations in metabolic processes and triggering DNA damage. The impact of exCh21 was also manifested in profound modifications in chromatin accessibility patterns. Moreover, we identified two signature metabolites, 5-oxo-ETE and Calcitriol, whose biosynthesis is affected by exCh21. Notably, supplementation with 5-oxo-ETE promoted DNA damage, in stark contrast to the protective effect elicited by Calcitriol against such damage. We also found that exCh21 disrupted cardiogenesis, and that this impairment could be mitigated through supplementation with Calcitriol. Specifically, the deleterious effects of 5-oxo-ETE unfolded in the form of DNA damage induction and the repression of cardiogenesis. On the other hand, Calcitriol emerged as a potent activator of its nuclear receptor VDR, fostering amplified binding to chromatin and subsequent facilitation of gene transcription. Our findings provide a comprehensive understanding of exCh21's metabolic implications within the context of Down syndrome, offering potential avenues for therapeutic interventions for Down syndrome treatment.

Engineered platforms for mimicking cardiac development and drug screening.

Congenital heart defects are associated with significant health challenges, demanding a deep understanding of the underlying biological mechanisms and, thus, better devices or platforms that can recapitulate human cardiac development. The discovery of human pluripotent stem cells has substantially reduced the dependence on animal models. Recent advances in stem cell biology, genetic editing, omics, microfluidics, and sensor technologies have further enabled remarkable progress in the development of in vitro platforms with increased fidelity and efficiency. In this review, we provide an overview of advancements in in vitro cardiac development platforms, with a particular focus on technological innovation. We categorize these platforms into four areas: two-dimensional solid substrate cultures, engineered substrate architectures that enhance cellular functions, cardiac organoids, and embryos/explants-on-chip models. We conclude by addressing current limitations and presenting future perspectives.

From phenotype to mechanism: Prenatal spectrum of NKAP mutation-related disorder and its pathogenesis inducing congenital heart disease.

NKAP mutations are associated with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD, MIM: #301039). Here, we elucidate the potential prenatal manifestation of NKAP mutation-associated disorder for the first time, alongside revealing the relationship between NKAP mutations and congenital heart defect (CHD) in the Chinese population. An NKAP mutation (NM_024528.4: c.988C>T, p.Arg330Cys) was identified in two foetuses presenting with CHD. Subsequent mechanistic exploration revealed a marked downregulation of NKAP transcription within HEK293T cells transfected with NKAP p.R330C. However, no significant change was observed at the protein level. Moreover, the mutation led to a dysregulation in the transcription of genes associated with cardiac morphogenesis, such as DHRS3, DNAH11 and JAG1. Additionally, our research determined that NKAP p.R330C affected Nkap protein intra-nuclear distribution, and binding with Hdac3. Summarily, our study strengthens NKAP mutations as a cause of CHD and prompts the reclassification of NKAP p.R330C as likely pathogenic, thereby establishing a prospective prenatal phenotypic spectrum that provides new insight into the prenatal diagnosis of CHD. Our findings also provide evidence of NKAP p.R330C pathogenicity and demonstrate the potential mechanism by which p.R330C dysregulates cardiac developmental gene transcription by altering Nkap intra-nuclear distribution and obstructing the interaction between Nkap and Hdac3, thereby leading to CHD.

Functions of cilia in cardiac development and disease.

Errors in embryonic cardiac development are a leading cause of congenital heart defects (CHDs), including morphological abnormalities of the heart that are often detected after birth. In the past few decades, an emerging role for cilia in the pathogenesis of CHD has been identified, but this topic still largely remains an unexplored area. Mouse forward genetic screens and whole exome sequencing analysis of CHD patients have identified enrichment for de novo mutations in ciliary genes or non-ciliary genes, which regulate cilia-related pathways, linking cilia function to aberrant cardiac development. Key events in cardiac morphogenesis, including left-right asymmetric development of the heart, are dependent upon cilia function. Cilia dysfunction during left-right axis formation contributes to CHD as evidenced by the substantial proportion of heterotaxy patients displaying complex CHD. Cilia-transduced signaling also regulates later events during heart development such as cardiac valve formation, outflow tract septation, ventricle development, and atrioventricular septa formation. In this review, we summarize the role of motile and non-motile (primary cilia) in cardiac asymmetry establishment and later events during heart development.

Cardiac development demystified by use of the HDBR atlas.

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.

Cardiac Metabolism.

The heart is composed of a heterogeneous mixture of cellular components perfectly intermingled and able to integrate common environmental signals to ensure proper cardiac function and performance. Metabolism defines a cell context-dependent signature that plays a critical role in survival, proliferation, or differentiation, being a recognized master piece of organ biology, modulating homeostasis, disease progression, and adaptation to tissue damage. The heart is a highly demanding organ, and adult cardiomyocytes require large amount of energy to fulfill adequate contractility. However, functioning under oxidative mitochondrial metabolism is accompanied with a concomitant elevation of harmful reactive oxygen species that indeed contributes to the progression of several cardiovascular pathologies and hampers the regenerative capacity of the mammalian heart. Cardiac metabolism is dynamic along embryonic development and substantially changes as cardiomyocytes mature and differentiate within the first days after birth. During early stages of cardiogenesis, anaerobic glycolysis is the main energetic program, while a progressive switch toward oxidative phosphorylation is a hallmark of myocardium differentiation. In response to cardiac injury, different signaling pathways participate in a metabolic rewiring to reactivate embryonic bioenergetic programs or the utilization of alternative substrates, reflecting the flexibility of heart metabolism and its central role in organ adaptation to external factors. Despite the well-established metabolic pattern of fetal, neonatal, and adult cardiomyocytes, our knowledge about the bioenergetics of other cardiac populations like endothelial cells, cardiac fibroblasts, or immune cells is limited. Considering the close intercellular communication and the influence of nonautonomous cues during heart development and after cardiac damage, it will be fundamental to better understand the metabolic programs in different cardiac cells in order to develop novel interventional opportunities based on metabolic rewiring to prevent heart failure and improve the limited regenerative capacity of the mammalian heart.

How best can we name the channels seen in the setting of deficient ventricular septation?

Surgical repair of channels between the ventricles is enhanced when the surgeon knows precisely where to place a patch, or baffle, so as to restore septal integrity. The paediatric cardiologist should provide the necessary information. Communication will be enhanced if the same words are used to account for the structures in question. Currently, however, the same term, namely "ventricular septal defect," is used to account for markedly different areas within the heart. Closure of perimembranous defects found in hearts with concordant or discordant ventriculo-arterial connections restores the integrity of the ventricular septum, at the same time separating the systemic and pulmonary blood streams. When both arterial trunks arise from the right ventricle, in contrast, the surgeon when placing a baffle so as to separate the blood streams, does not close the channel most frequently described as the "ventricular septal defect." In this review, we show that the perimembranous lesions as found in hearts with concordant or discordant ventriculo-arterial connections are the right ventricular entrances to the areas subtended beneath the hinges of the leaflets of the aortic or pulmonary valves. When both arterial trunks arise from the right ventricle, and the channel between the ventricles is directly subaortic, then the channel termed the "ventricular septal defect" is the left ventricular entrance to the comparable space subtended beneath the aortic root. We argue that recognition of these fundamental anatomical differences enhances the appreciation of the underlying morphology of the various lesions that reflect transfer, during cardiac development, of the aortic root from the morphologically right to the morphologically left ventricle.

Sex-Based Mechanisms of Cardiac Development and Function: Applications for Induced-Pluripotent Stem Cell Derived-Cardiomyocytes.

Males and females exhibit intrinsic differences in the structure and function of the heart, while the prevalence and severity of cardiovascular disease vary in the two sexes. However, the mechanisms of this sex-based dimorphism are yet to be elucidated. Sex chromosomes and sex hormones are the main contributors to sex-based differences in cardiac physiology and pathophysiology. In recent years, the advances in induced pluripotent stem cell-derived cardiac models and multi-omic approaches have enabled a more comprehensive understanding of the sex-specific differences in the human heart. Here, we provide an overview of the roles of these two factors throughout cardiac development and explore the sex hormone signaling pathways involved. We will also discuss how the employment of stem cell-based cardiac models and single-cell RNA sequencing help us further investigate sex differences in healthy and diseased hearts.

Cardiac Development and Factors Influencing the Development of Congenital Heart Defects (CHDs): Part I.

The traditional description of cardiac development involves progression from a cardiac crescent to a linear heart tube, which in the phase of transformation into a mature heart forms a cardiac loop and is divided with the septa into individual cavities. Cardiac morphogenesis involves numerous types of cells originating outside the initial cardiac crescent, including neural crest cells, cells of the second heart field origin, and epicardial progenitor cells. The development of the fetal heart and circulatory system is subject to regulatation by both genetic and environmental processes. The etiology for cases with congenital heart defects (CHDs) is largely unknown, but several genetic anomalies, some maternal illnesses, and prenatal exposures to specific therapeutic and non-therapeutic drugs are generally accepted as risk factors. New techniques for studying heart development have revealed many aspects of cardiac morphogenesis that are important in the development of CHDs, in particular transposition of the great arteries.

miR-1 as a Key Epigenetic Regulator in Early Differentiation of Cardiac Sinoatrial Region.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

Ventricular anisotropic deformation and contractile function of the developing heart of zebrafish in vivo.

BACKGROUND: The developing zebrafish ventricle generates higher intraventricular pressure (IVP) with increasing stroke volume and cardiac output at different developmental stages to meet the metabolic demands of the rapidly growing embryo (Salehin et al. Ann Biomed Eng, 2021;49(9): 2080-2093). To understand the changing role of the developing embryonic heart, we studied its biomechanical characteristics during in vivo cardiac cycles. By combining changes in wall strains and IVP measurements, we assessed ventricular wall stiffness during diastolic filling and the ensuing systolic IVP-generation capacity during 3-, 4-, and 5-day post fertilization (dpf). We further examined the anisotropy of wall deformation, in different regions within the ventricle, throughout a complete cardiac cycle. RESULTS: We found the ventricular walls grow increasingly stiff during diastolic filling with a corresponding increase in IVP-generation capacity from 3- to 4- and 5-dpf groups. In addition, we found the corresponding increasing level of anisotropic wall deformation through cardiac cycles that favor the latitudinal direction, with the most pronounced found in the equatorial region of the ventricle. CONCLUSIONS: From 3- to 4- and 5-dpf groups, the ventricular wall myocardium undergoes increasing level of anisotropic deformation. This, in combination with the increasing wall stiffness and IVP-generation capacity, allows the developing heart to effectively pump blood to meet the rapidly growing embryo's needs.

Quantitative proteomics reveals PPAR signaling pathway regulates the cardiomyocyte activity of neonatal mouse heart.

Cardiovascular diseases (CVDs) are among the most morbid and deadly types of diseases worldwide, while the existing therapeutic approaches all have their limitations. Mouse heart undergoes a very complex postnatal developmental process, including the 1-week window in which cardiomyocytes (CMs) maintain relatively high cell activity. The underlying mechanism provides an attractive direction for CVDs treatment. Herein, we collected ventricular tissues from mice of different ages from E18.5D to P8W and performed iTRAQ-based quantitative proteomics to characterize the atlas of cardiac development. A total of 3422 proteins were quantified at all selected time points, revealing critical proteomic changes related to cardiac developmental events such as the metabolic transition from glycolysis to beta-oxidation. A cluster of significantly dysregulated proteins containing proteins that have already been reported to be associated with cardiac regeneration (Erbb2, Agrin, and Hmgb) was identified. Meanwhile, the peroxisome proliferator-activated receptor (PPAR) signaling pathway (Cpt1α, Hmgcs2, Plin2, and Fabp4) was also found specifically enriched. We further revealed that bezafibrate, a pan-activator of PPAR signaling pathway markedly enhanced H9C2 cardiomyocyte activity via enhancing Cpt1α expression. This work provides new hint that activation of PPAR signaling pathway could potentially be a therapeutic strategy for the treatment of CVDs.

Single cell RNA sequencing approaches to cardiac development and congenital heart disease.

The development of single cell RNA sequencing technologies has accelerated the ability of scientists to understand healthy and disease states of the cardiovascular system. Congenital heart defects occur in approximately 40,000 births each year and 1 out of 4 children are born with critical congenital heart disease requiring surgical interventions and a lifetime of monitoring. An understanding of how the normal heart develops and how each cell contributes to normal and pathological anatomy is an important goal in pediatric cardiovascular research. Single cell sequencing has provided the tools to increase the ability to discover rare cell types and novel genes involved in normal cardiac development. Knowledge of gene expression of single cells within cardiac tissue has contributed to the understanding of how each cell type contributes to the anatomic structures of the heart. In this review, we summarize how single cell RNA sequencing has been utilized to understand cardiac developmental processes and congenital heart disease. We discuss the advantages and disadvantages of whole cell versus single nuclei RNA sequencing and describe the approaches to analyze the interactomes, transcriptomes, and differentiation trajectory from single cell data. We summarize the currently available single cell RNA sequencing technologies and technical aspects of performing single cell analysis and how to overcome common obstacles. We also review data from the recently published human and mouse fetal heart atlases and advancements that have occurred within the field due to the application of these single cell tools. Finally we highlight the potential for single cell technologies to uncover novel mechanisms of disease pathogenesis by leveraging findings from genome wide association studies.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes.

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.

The G4 resolvase RHAU regulates ventricular trabeculation and compaction through transcriptional and post-transcriptional mechanisms.

The G-quadruplex (G4) resolvase RNA helicase associated with AU-rich element (RHAU) possesses the ability to unwind G4 structures in both DNA and RNA molecules. Previously, we revealed that RHAU plays a critical role in embryonic heart development and postnatal heart function through modulating mRNA translation and stability. However, whether RHAU functions to resolve DNA G4 in the regulation of cardiac physiology is still elusive. Here, we identified a phenotype of noncompaction cardiomyopathy in cardiomyocyte-specific Rhau deletion mice, including such symptoms as spongiform cardiomyopathy, heart dilation, and death at young ages. We also observed reduced cardiomyocyte proliferation and advanced sarcomere maturation in Rhau mutant mice. Further studies demonstrated that RHAU regulates the expression levels of several genes associated with ventricular trabeculation and compaction, including the Nkx2-5 and Hey2 that encode cardiac transcription factors of NKX2-5 and Hey2, and the myosin heavy chain 7 (Myh7) whose protein product is MYH7. While RHAU modulates Nkx2-5 mRNA and Hey2 mRNA at the post-transcriptional level, we uncovered that RHAU facilitates the transcription of Myh7 through unwinding of the G4 structures in its promoter. These findings demonstrated that RHAU regulates ventricular chamber development through both transcriptional and post-transcriptional mechanisms. These results contribute to a knowledge base that will help to understand the pathogenesis of diseases such as noncompaction cardiomyopathy.

Llgl1 regulates zebrafish cardiac development by mediating Yap stability in cardiomyocytes.

The Hippo-Yap pathway regulates multiple cellular processes in response to mechanical and other stimuli. In Drosophila, the polarity protein Lethal (2) giant larvae [L(2)gl], negatively regulates Hippo-mediated transcriptional output. However, in vertebrates, little is known about its homolog Llgl1. Here, we define a novel role for vertebrate Llgl1 in regulating Yap stability in cardiomyocytes, which impacts heart development. In contrast to the role of Drosophila L(2)gl, Llgl1 depletion in cultured rat cardiomyocytes decreased Yap protein levels and blunted target gene transcription without affecting Yap transcript abundance. Llgl1 depletion in zebrafish resulted in larger and dysmorphic cardiomyocytes, pericardial effusion, impaired blood flow and aberrant valvulogenesis. Cardiomyocyte Yap protein levels were decreased in llgl1 morphants, whereas Notch, which is regulated by hemodynamic forces and participates in valvulogenesis, was more broadly activated. Consistent with the role of Llgl1 in regulating Yap stability, cardiomyocyte-specific overexpression of Yap in Llgl1-depleted embryos ameliorated pericardial effusion and restored blood flow velocity. Altogether, our data reveal that vertebrate Llgl1 is crucial for Yap stability in cardiomyocytes and its absence impairs cardiac development.