Cardiac Gene Regulatory Network Research Articles

An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility.

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)) and one downstream (RE(down)) of Tbx5. In both lines, we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial Tbx5 expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in Tbx5RE(int)KO cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in Tbx5RE(int)KO mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

An atlas of transcribed human cardiac promoters and enhancers reveals an important role of regulatory elements in heart failure.

A deeper knowledge of the dynamic transcriptional activity of promoters and enhancers is needed to improve mechanistic understanding of the pathogenesis of heart failure and heart diseases. In this study, we used cap analysis of gene expression (CAGE) to identify and quantify the activity of transcribed regulatory elements (TREs) in the four cardiac chambers of 21 healthy and ten failing adult human hearts. We identified 17,668 promoters and 14,920 enhancers associated with the expression of 14,519 genes. We showed how these regulatory elements are alternatively transcribed in different heart regions, in healthy versus failing hearts and in ischemic versus non-ischemic heart failure samples. Cardiac-disease-related single-nucleotide polymorphisms (SNPs) appeared to be enriched in TREs, potentially affecting the allele-specific transcription factor binding. To conclude, our open-source heart CAGE atlas will serve the cardiovascular community in improving the understanding of the role of the cardiac gene regulatory networks in cardiovascular disease and therapy.

Abstract MP208: A Shared Fog2 / Tbx5-dependent Gene Regulatory Network Identified By Transcription Factor-dependent Non-coding RNA Profiling Modulates Cardiac Rhythm

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting over 33 million individuals throughout the world. AF is highly heritable and recent genome-wide association studies (GWAS) have cumulatively identified over 100 loci associated with AF risk. Genome-wide association studies (GWAS) often identify transcription factor (TF) loci in association with complex human diseases, implying that a significant transcriptional component underlies human disease risk and etiology. The transcription factors ZFPM2 (FOG2), GATA4, and TBX5 have all been implicated in human AF risk by genetic studies. We hypothesized that FOG2, GATA4, and TBX5 functionally interact to regulate a gene regulatory network essential for atrial rhythm control. We generated a novel mouse model of spontaneous AF based on FOG2 overexpression. FOG2 ChIP-seq identified FOG2 genomic localization at loci co-occupied by GATA4, a known FOG2 binding partner. However, we found that FOG2 OE caused gene expression alterations that correlated more highly with TBX5-dependent rather than GATA4-dependent gene expression, including a module of calcium handling genes required for atrial rhythm homeostasis. We applied TF-dependent non-coding transcriptional profiling to examine the FOG2 dependent atrial GRN, which identified 805 candidate regulatory regions with accessible chromatin and FOG2 dependent ncRNAs. TBX5 removal and FOG2 OE caused highly correlated dysregulation of ncRNA expression at open chromatin regions genome-wide, suggesting a functional interaction between TBX5 and FOG2. Furthermore, FOG2 OE only affected enhancer activity by altered ncRNA abundance at locations of TBX5 co-binding. The shared TBX5/FOG2 genomic interaction predicted a potential genetic interaction, and we found that cardiac rhythm abnormalities caused by Tbx5 haploinsufficiency were rescued by Fog2 haploinsufficiency. Taken together, TF-dependent ncRNA-profiling revealed an interconnected cardiac rhythm gene regulatory network (GRN) between FOG2, TBX5 and GATA4. These data nominate a specific model in which FOG2 is recruited by GATA4 to modulate a co-bound TBX5-dependent atrial gene regulatory network for calcium handling and atrial rhythm homeostasis.

Towards spatio-temporally resolved developmental cardiac gene regulatory networks in zebrafish.

Heart formation in the zebrafish involves a rapid, complex series of morphogenetic events in three-dimensional space that spans cardiac lineage specification through to chamber formation and maturation. This process is tightly orchestrated by a cardiac gene regulatory network (GRN), which ensures the precise spatio-temporal deployment of genes critical for heart formation. Alterations of the timing or spatial localisation of gene expression can have a significant impact in cardiac ontogeny and may lead to heart malformations. Hence, a better understanding of the cellular and molecular basis of congenital heart disease relies on understanding the behaviour of cardiac GRNs with precise spatiotemporal resolution. Here, we review the recent technical advances that have expanded our capacity to interrogate the cardiac GRN in zebrafish. In particular, we focus on studies utilising high-throughput technologies to systematically dissect gene expression patterns, both temporally and spatially during heart development.

Single-Cell Transcriptomic Analysis of Cardiac Progenitor Differentiation

Emerging single-cell RNA sequencing technologies hold great promises to boost our understanding of the heterogeneity and molecular regulation of diverse cell phenotypes during organ development. In this review, we aimed at summarizing recent advances in employing single-cell transcriptomic analysis to depict the landscape of embryonic heart development, in particular, focusing on cardiac progenitor (CP) differentiation. Recent studies unbiasedly cataloged and characterized cardiac cell types in the spatial and temporal resolution during early heart development. Pseudo-time analysis revealed a temporal continuum of the differentiation progress from embryonic day (E) 6.5 to E9.5, implicating early cardiac lineage restriction during mouse gastrulation. First and second heart field (FHF and SHF) CPs adopted different differentiation strategies and underwent distinct transcriptional regulation. Collectively, the comprehensive molecular atlases yield a rich resource for identification of the key cardiac regulators and signaling molecules within the key cardiac gene regulatory network (GRN) governing cardiac cell fate determinations. This review offers insights into the exquisite process and its regulation of CP differentiation at single-cell resolution. As single-cell technologies continuously grow and evolve, computational integration of multimodal single-cell data with well-designed experimental validation promises to further delineate molecular basis in deploying cardiac progenitors of distinct sources with anatomical information.

Abstract 281: Single Cell Analysis of Postnatal Heart Development and Disease

A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell-type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-Seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-Seq overcomes the technical challenge of isolating intact single cell from complex tissues including the maturing mammalian hearts, reduces biased recovery of easily dissociated cell types and minimizes aberrant gene expression during the whole-cell dissociation. We have recently applied sNucDrop-Seq, a droplet microfluidics-based massively parallel snRNA-Seq method, to investigate the transcriptional landscape of postnatal mouse hearts in both healthy and mitochondrial disease states. By profiling the transcriptome of nearly 20,000 nuclei, we identified major and rare cardiac cell types and revealed significant cellular heterogeneity in the postnatal developing heart. When applied to a mouse model of mitochondrial cardiomyopathy, we uncovered profound cell type-specific modifications of the cardiac transcriptional landscape at single-nucleus resolution. Here, we expanded these earlier studies and used our dataset to further decipher the cardiac cell type-specific gene regulatory networks. Our analysis reveals novels insights into the key nodes of gene networks that control the postnatal development and disease-associated changes of different cardiac cell types. Our ongoing work is using genetic mouse models to validate these findings from single cell analysis.

Abstract 705: Synergistic Activation of the Cardiac Enhancer Landscape During Cardiac Reprogramming

Direct cardiac reprogramming of fibroblasts to cardiomyocytes is an attractive therapeutic strategy to restore cardiac function following injury. The cardiac transcription factors Gata4, Mef2c, and Tbx5 are sufficient to directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the addition of Hand2 and Akt1. However, the mechanisms by which these transcription factors orchestrate this cell fate conversion remains elusive. To understand the mechanistic basis of cardiac reprogramming, we performed a genome-wide analysis of cardiogenic transcription factor binding sites and enhancer activation throughout cardiac reprogramming. We found that cardiogenic transcription factors act cooperatively by co-occupying regulatory elements enriched with Mef2 binding sites during cardiac reprogramming. Importantly, these transcription factors when overexpressed in isolation are incapable of activating reprogramming enhancer elements. Further, we discovered that the early reprogramming enhancer landscape most closely resembles that of the neonatal heart. Acquisition of enhancers associated with cardiac maturation occurred at later stages in reprogramming, with addition of Hand2 and Akt1 augmenting activation of this more mature enhancer landscape. Additionally, we constructed a cardiac reprogramming gene regulatory network which demonstrated downregulation of the EGF receptor signaling pathway. We found that inhibition of EGF receptor signaling augments reprogramming toward the cardiac phenotype. Our findings demonstrate that cardiac reprogramming is coordinated by synergistic transcriptional activation across a broad landscape of cardiac enhancers and define the epigenetic landscape of the cardiac phenotype.

Cardiac Reprogramming Factors Synergistically Activate Genome-wide Cardiogenic Stage-Specific Enhancers.

The cardiogenic transcription factors (TFs) Mef2c, Gata4, and Tbx5 can directly reprogram fibroblasts to induced cardiac-like myocytes (iCLMs), presenting a potential source of cells for cardiac repair. While activity of these TFs is enhanced by Hand2 and Akt1, their genomic targets and interactions during reprogramming are not well studied. We performed genome-wide analyses of cardiogenic TF binding and enhancer profiling during cardiac reprogramming. We found that these TFs synergistically activate enhancers highlighted by Mef2c binding sitesand that Hand2 and Akt1 coordinately recruit other TFs to enhancer elements. Intriguingly, these enhancer landscapes collectively resemble patterns of enhancer activation during embryonic cardiogenesis. We further constructed a cardiac reprogramming gene regulatory network and found repression of EGFR signaling pathway genes. Consistently, chemical inhibition of EGFR signaling augmented reprogramming. Thus, by defining epigenetic landscapes these findings reveal synergistic transcriptional activation across a broad landscape of cardiac enhancers and key signaling pathways that govern iCLM reprogramming.

Single-nucleus transcriptomic survey of cell diversity and functional maturation in postnatal mammalian hearts.

A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-seq overcomes the technical challenge of isolating intact single cells from complex tissues, including the maturing mammalian hearts; reduces biased recovery of easily dissociated cell types; and minimizes aberrant gene expression during the whole-cell dissociation. Here we applied sNucDrop-seq, a droplet microfluidics-based massively parallel snRNA-seq method, to investigate the transcriptional landscape of postnatal maturing mouse hearts in both healthy and disease states. By profiling the transcriptome of nearly 20,000 nuclei, we identified major and rare cardiac cell types and revealed significant heterogeneity of cardiomyocytes, fibroblasts, and endothelial cells in postnatal developing hearts. When applied to a mouse model of pediatric mitochondrial cardiomyopathy, we uncovered profound cell type-specific modifications of the cardiac transcriptional landscape at single-nucleus resolution, including changes of subtype composition, maturation states, and functional remodeling of each cell type. Furthermore, we employed sNucDrop-seq to decipher the cardiac cell type-specific gene regulatory network (GRN) of GDF15, a heart-derived hormone and clinically important diagnostic biomarker of heart disease. Together, our results present a rich resource for studying cardiac biology and provide new insights into heart disease using an approach broadly applicable to many fields of biomedicine.

Abstract 371: Cardiac Endocrinology: Biology of a Heart-derived Hormone That Regulates Body Growth

The endocrine system is crucial for maintaining whole-body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than ANP and BNP discovered over 30 years ago. We recently identified growth differentiation factor 15 (GDF15) as a heart-derived hormone that regulates body growth. Pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. Blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart-derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive. In addition, we used massively parallel single-nucleus RNA-Seq to decipher cardiac cell type specific gene regulatory network that induces GDF15 transcription in heart disease condition. We further determined how GDF15 precursor is processed into mature hormone. Together these studies reveal a new endocrine mechanism (cardiac endocrinology) by which the heart coordinates cardiac function and body growth.

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

Prediction and validation of protein-protein interactors from genome-wide DNA-binding data using a knowledge-based machine-learning approach.

The ability to accurately predict the DNA targets and interacting cofactors of transcriptional regulators from genome-wide data can significantly advance our understanding of gene regulatory networks. NKX2-5 is a homeodomain transcription factor that sits high in the cardiac gene regulatory network and is essential for normal heart development. We previously identified genomic targets for NKX2-5 in mouse HL-1 atrial cardiomyocytes using DNA-adenine methyltransferase identification (DamID). Here, we apply machine learning algorithms and propose a knowledge-based feature selection method for predicting NKX2-5 protein : protein interactions based on motif grammar in genome-wide DNA-binding data. We assessed model performance using leave-one-out cross-validation and a completely independent DamID experiment performed with replicates. In addition to identifying previously described NKX2-5-interacting proteins, including GATA, HAND and TBX family members, a number of novel interactors were identified, with direct protein : protein interactions between NKX2-5 and retinoid X receptor (RXR), paired-related homeobox (PRRX) and Ikaros zinc fingers (IKZF) validated using the yeast two-hybrid assay. We also found that the interaction of RXRα with NKX2-5 mutations found in congenital heart disease (Q187H, R189G and R190H) was altered. These findings highlight an intuitive approach to accessing protein–protein interaction information of transcription factors in DNA-binding experiments.

The Function of the MEF2 Family of Transcription Factors in Cardiac Development, Cardiogenomics, and Direct Reprogramming.

Proper formation of the mammalian heart requires precise spatiotemporal transcriptional regulation of gene programs in cardiomyocytes. Sophisticated regulatory networks have evolved to not only integrate the activities of distinct transcription factors to control tissue-specific gene programs but also, in many instances, to incorporate multiple members within these transcription factor families to ensure accuracy and specificity in the system. Unsurprisingly, perturbations in this elaborate transcriptional circuitry can lead to severe cardiac abnormalities. Myocyte enhancer factor–2 (MEF2) transcription factor belongs to the evolutionarily conserved cardiac gene regulatory network. Given its central role in muscle gene regulation and its evolutionary conservation, MEF2 is considered one of only a few core cardiac transcription factors. In addition to its firmly established role as a differentiation factor, MEF2 regulates wide variety of, sometimes antagonistic, cellular processes such as cell survival and death. Vertebrate genomes encode multiple MEF2 family members thereby expanding the transcriptional potential of this core transcription factor in the heart. This review highlights the requirement of the MEF2 family and their orthologs in cardiac development in diverse animal model systems. Furthermore, we describe the recently characterized role of MEF2 in direct reprogramming and genome-wide cardiomyocyte gene regulation. A thorough understanding of the regulatory functions of the MEF2 family in cardiac development and cardiogenomics is required in order to develop effective therapeutic strategies to repair the diseased heart.

CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis

Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.

Myocardin-related transcription factors are required for cardiac development and function

Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the developing or adult heart has not been explored. Through cardiac-specific deletion of MRTF alleles in mice, we show that either MRTF-A or MRTF-B is dispensable for cardiac development and function, whereas deletion of both MRTF-A and MRTF-B causes a spectrum of structural and functional cardiac abnormalities. Defects observed in MRTF-A/B null mice ranged from reduced cardiac contractility and adult onset heart failure to neonatal lethality accompanied by sarcomere disarray. RNA-seq analysis on neonatal hearts identified the most altered pathways in MRTF double knockout hearts as being involved in cytoskeletal organization. Together, these findings demonstrate redundant but essential roles of the MRTFs in maintenance of cardiac structure and function and as indispensible links in cardiac cytoskeletal gene regulatory networks.

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

AimHeart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis.Methods and resultsWe annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease.ConclusionThese findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.

Novel and functional variants within the TBX18 gene promoter in ventricular septal defects

Congenital heart disease (CHD) is the most common birth defect in humans. Genetic causes for CHD remain largely unknown. T-box transcription factor 18 (TBX18) gene is expressed in the developing heart, including myocardium of the left ventricle and interventricular septum. Epicardial cells expressing TBX18 gene contribute to the cardiac fibroblast and smooth muscle cells. We speculated that the DNA sequence variants (DSVs) within TBX18 gene promoter may mediate CHD development by affecting TBX18 levels and the cardiac gene regulatory network. In this study, we genetically and functionally analyzed the TBX18 gene promoter in patients with ventricular septal defects (VSD) (n = 326) and ethnic-matched healthy controls (n = 327). Three novel heterozygous DSVs (g.85474435del, g.85474418C>T, and g.85473965C>G) and one single nucleotide polymorphism (g.85474871C>T, rs77693245) were identified in VSD patients, but none in the controls. Functional analysis revealed that the DSVs (g.85474871C>T, g.85474435del, and g.85473965C>G) significantly decreased the transcriptional activities of the TBX18 gene promoter. The effect of DSV (g.85474418C>T) on the TBX18 gene promoter was marginal, but not significant. Therefore, the DSVs within the TBX18 gene promoter identified in VSD patients may be involved in the VSD etiology.

Two novel and functional DNA sequence variants within an upstream enhancer of the human NKX2-5 gene in ventricular septal defects

Mortality in patients with congenital heart disease (CHD) is significantly increased even with successful surgeries. The main causes are late cardiac complications, such as heart failure and arrhythmia, probably due to genetic defects. To date, genetic causes for CHD remain largely unknown. NKX2-5 gene encodes a highly conserved homeobox transcription factor, which is essential to the heart development in embryos and cardiac function in adults. Mutations in NKX2-5 gene have been implicated in diverse types of CHD, including ventricular septal defect (VSD). As NKX2-5 is a dosage-sensitive regulator, we have speculated that changed NKX2-5 levels may mediate CHD development by influencing cardiac gene regulatory network. In previous studies, we have analyzed the NKX2-5 gene promoter and a proximal enhancer in VSD patients. In the present study, we further genetically and functionally analyzed an upstream enhancer of the NKX2-5 gene in large cohorts of VSD patients (n=340) and controls (n=347). Two novel heterozygous DNA sequence variants (DSVs), g.17483576C>G and g.17483564C>T, were identified in three VSD patients, but none in controls. Functionally, these two DSVs significantly decreased the activity of the enhancer (P<0.01). Another novel heterozygous DSV, g.17483557Ins, was found in both VSD patients and controls with similar frequencies (P>0.05). Taken together, our data suggested that the DSVs within the upstream enhancer of the NKX2-5 gene may contribute to a small number of VSD. Therefore, genetic studies of CHD may provide insight into designing novel therapies for adult CHD patients.

A boolean model of the cardiac gene regulatory network determining first and second heart field identity.

Two types of distinct cardiac progenitor cell populations can be identified during early heart development: the first heart field (FHF) and second heart field (SHF) lineage that later form the mature heart. They can be characterized by differential expression of transcription and signaling factors. These regulatory factors influence each other forming a gene regulatory network. Here, we present a core gene regulatory network for early cardiac development based on published temporal and spatial expression data of genes and their interactions. This gene regulatory network was implemented in a Boolean computational model. Simulations reveal stable states within the network model, which correspond to the regulatory states of the FHF and the SHF lineages. Furthermore, we are able to reproduce the expected temporal expression patterns of early cardiac factors mimicking developmental progression. Additionally, simulations of knock-down experiments within our model resemble published phenotypes of mutant mice. Consequently, this gene regulatory network retraces the early steps and requirements of cardiogenic mesoderm determination in a way appropriate to enhance the understanding of heart development.