ABSTRACT
Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7â bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Gene Expression Regulation, Developmental , Heart , Promoter Regions, Genetic , Transcription Factors , Animals , Promoter Regions, Genetic/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Heart/embryology , Transcription Factors/metabolism , Transcription Factors/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Mutation/genetics , Embryonic Development/genetics , Humans , Transcription, Genetic , Repressor Proteins , Trans-ActivatorsABSTRACT
BACKGROUND: Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocyte cells. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. METHODS: CMs were derived from hiPSC (hi-PCS-CM) using different concentrations of RA (Control without RA, LRA with 0.05µM and HRA with 0.1 µM) between day 3-6 of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling hiPSC-CM at high cell density in a low collagen hydrogel. RESULTS: In the HRA group, hiPSC-CMs exhibited highest expression of contractile proteins MYH6, MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are marker genes for left ventricular CMs, was also the highest in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was functionally more similar to the left ventricle. RNAsequencing revealed that the heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. CONCLUSION: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHTs with a phenotype similar to that of the left ventricle or right ventricle.
Subject(s)
Cell Differentiation , Heart Ventricles , Induced Pluripotent Stem Cells , Myocytes, Cardiac , Tretinoin , Humans , Tretinoin/pharmacology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Cell Differentiation/drug effects , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Heart Ventricles/cytology , Heart Ventricles/metabolism , Myosin Heavy Chains/metabolism , Myosin Heavy Chains/genetics , Cardiac Myosins/metabolism , Cardiac Myosins/genetics , Tissue Engineering/methods , Homeobox Protein Nkx-2.5/metabolism , Homeobox Protein Nkx-2.5/genetics , T-Box Domain Proteins/metabolism , T-Box Domain Proteins/geneticsABSTRACT
The prevalence of dilated cardiomyopathy (DCM) is increasing globally, highlighting the need for innovative therapeutic approaches to prevent its onset. In this study, we examined the energetic and epigenetic distinctions between dilated and non-dilated human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and assessed the effects of class I and II HDAC inhibitors (HDACi) on these cells and their cardiomyogenic differentiation. Cells were isolated from myocardium biopsies using explant outgrowth methods. Mitochondrial and histone deacetylase activities, ATP levels, cardiac transcription factors, and structural proteins were assessed using flow cytometry, PCR, chemiluminescence, Western blotting, and immunohistochemistry. The data suggest that the tested HDAC inhibitors improved acetylation and enhanced the energetic status of both types of cells, with significant effects observed in dilated myocardium-derived hmMSCs. Additionally, the HDAC inhibitors activated the cardiac transcription factors Nkx2-5, HOPX, GATA4, and Mef2C, and upregulated structural proteins such as cardiac troponin T and alpha cardiac actin at both the protein and gene levels. In conclusion, our findings suggest that HDACi may serve as potential modulators of the energetic status and cardiomyogenic differentiation of human heart hmMSCs. This avenue of exploration could broaden the search for novel therapeutic interventions for dilated cardiomyopathy, ultimately leading to improvements in heart function.
Subject(s)
Cardiomyopathy, Dilated , Cell Differentiation , Histone Deacetylase Inhibitors , Mesenchymal Stem Cells , Humans , Histone Deacetylase Inhibitors/pharmacology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/cytology , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/pathology , Cell Differentiation/drug effects , Myocardium/cytology , Myocardium/metabolism , Myocardium/pathology , Histone Deacetylases/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , MEF2 Transcription Factors/metabolism , MEF2 Transcription Factors/genetics , Homeobox Protein Nkx-2.5/metabolism , Homeobox Protein Nkx-2.5/genetics , Acetylation/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Cells, CulturedABSTRACT
Heart failure is a serious medical condition with a poor prognosis. Current treatments can only help manage the symptoms and slow the progression of heart failure. However, there is currently no cure to prevent and reverse cardiac remodeling. Transcription factors are in a central role in various cellular processes, and in the heart, GATA4 and NKX2-5 transcription factors mediate hypertrophic responses and remodeling. We have identified compounds that modulate the synergistic interaction of GATA4 and NKX2-5 and shown that the most promising compound (1, 3i-1000) is cardioprotective in vitro and in vivo. However, direct evidence of its binding site and mechanism of action has not been available. Due to the disordered nature of transcription factors, classical target engagement approaches cannot be utilized. Here, we synthesized a small-molecule ligand-binding pulldown probe of compound 1 to utilize affinity chromatography alongside CETSA, AlphaScreen, and molecular modeling to study ligand binding. These results provide the first evidence of direct physical binding of compound 1 selectively to GATA4. While developing drugs that target transcription factors presents challenges, advances in technologies and knowledge of intrinsically disordered proteins enable the identification of small molecules that can selectively target transcription factors.
Subject(s)
Heart Failure , Transcription Factors , Humans , Homeobox Protein Nkx-2.5/metabolism , Ligands , Transcription Factors/metabolism , Chromatography, Affinity , GATA4 Transcription Factor/metabolism , Homeodomain Proteins/metabolismABSTRACT
NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-ß and further enhanced by hypoxia. The effect of TGF-ß was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.
Subject(s)
Homeobox Protein Nkx-2.5 , Muscle, Smooth, Vascular , Vascular Remodeling , Animals , Mice , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Humans , Vascular Remodeling/genetics , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Male , Scleroderma, Systemic/pathology , Scleroderma, Systemic/complications , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/genetics , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Pulmonary Arterial Hypertension/metabolism , Pulmonary Arterial Hypertension/genetics , Pulmonary Arterial Hypertension/pathology , Pulmonary Arterial Hypertension/etiology , Female , Transforming Growth Factor beta/metabolism , Disease Models, Animal , Cell Proliferation/genetics , Middle Aged , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/pathologyABSTRACT
The transcription factor HAND1 is a critical regulator of cardiac development which is expressed in sub-populations of cardiac progenitors and cardiomyocytes. The transcription factor NKX2-5, in contrast, is expressed more widely in cardiac cells. Here we report the generation of a dual reporter hESC line where the expression of these genes can be simultaneously measured, enabling lineage analysis as well as studies of HAND1 and NKX2-5 gene regulation and protein function. This tool will have wide utility particularly for research on developmental biology and disease modelling.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Homeobox Protein Nkx-2.5 , Homeodomain Proteins , Human Embryonic Stem Cells , Transcription Factors , Humans , Homeobox Protein Nkx-2.5/metabolism , Homeobox Protein Nkx-2.5/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Genes, Reporter , Cell Differentiation , Cell LineageABSTRACT
Mesenchymal stem cells (MSCs) treatment has been widely explored as a therapy for myocardial infarction, peripheral ischemic vascular diseases, dilated cardiomyopathy, and pulmonary hypertension. Latest in vitro studies suggest that MSCs can differentiate into contractile cardiomyocytes. One of the best-characterized MSCs products are MSCs-derived extracellular vesicles (EVs). EVs are crucial paracrine effectors of MSCs. Based on previous works, paracrine effects of MSCs play a primary role in the regenerative ability. Hence, in the current paper, we focused our attention on an alternative approach, exploiting products derived from human dental pulp stem cells (hDPSCs) rather than MSCs themselves, which may denote a cost-effective and safer approach. The focus has been on EVs and the bioactive molecules they contain to evaluate their ability to influence the differentiation process toward cardiomyogenic lineage. The expression of GATA4, ACTC1, CX43, and Nkx2.5 was evaluated using Immunofluorescence, real time-PCR, and Western blotting analyses. Furthermore, the expression profiling analysis of the microRNA hsa-miR-200c-3p, targeting the GATA4 gene, was studied. The hsa-miR-200c-3p was found significantly down-regulated in both c-hDPSCs + EVs-hDPSCs and c-hDPSCs + EVs-HL-1 compared to untreated c-hDPSCs underlying a possible epigenetic mechanism behind the prevalent up-regulation of its targeted GATA4 gene. The aim of the present work was to develop an in vitro model of hDPSCs able to differentiate into cardiomyocytes in order to investigate the role of EVs derived from hDPSCs and derived from HL-1 cardiomyocyte cell line in modulating the differentiation process toward cardiomyogenic lineage.
Subject(s)
Cell Differentiation , Dental Pulp , Extracellular Vesicles , Mesenchymal Stem Cells , MicroRNAs , Myocytes, Cardiac , Regeneration , Humans , Extracellular Vesicles/metabolism , Extracellular Vesicles/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , MicroRNAs/genetics , MicroRNAs/metabolism , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Dental Pulp/cytology , Dental Pulp/metabolism , Regeneration/physiology , Regeneration/genetics , Homeobox Protein Nkx-2.5/metabolism , Homeobox Protein Nkx-2.5/genetics , GATA4 Transcription Factor/metabolism , GATA4 Transcription Factor/genetics , Connexin 43/metabolism , Connexin 43/genetics , Cells, CulturedABSTRACT
The transcription factor NKX2-5 is a highly conserved master regulator of heart development which is widely expressed in cardiac progenitors and cardiomyocytes. Fluorescent reporters of NKX2-5 that minimally perturb normal protein expression can enable the identification, quantification and isolation of NKX2-5-expressing cells in a normal physiological state. Here we report the generation of two new hESC lines with eGFP inserted upstream (5') or downstream (3') of NKX2-5, linked by a cleavable T2A peptide. These complementary reporters produce a robust fluorescent signal in cardiac cells and have wide utility particularly for research on developmental biology and disease modelling.
Subject(s)
Human Embryonic Stem Cells , Humans , Human Embryonic Stem Cells/metabolism , Cell Differentiation , Embryonic Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Cell Line , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolismABSTRACT
Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis.
Subject(s)
Cardiovascular Diseases , Humans , Cardiovascular Diseases/genetics , Genetic Predisposition to Disease , Genome-Wide Association Study , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Myocytes, Cardiac/metabolism , Polymorphism, Single Nucleotide , Regulatory Sequences, Nucleic Acid , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Congenital heart disease often arises from perturbations of transcription factors (TFs) that guide cardiac development. ISLET1 (ISL1) is a TF that influences early cardiac cell fate, as well as differentiation of other cell types including motor neuron progenitors (MNPs) and pancreatic islet cells. While lineage specificity of ISL1 function is likely achieved through combinatorial interactions, its essential cardiac interacting partners are unknown. By assaying ISL1 genomic occupancy in human induced pluripotent stem cell-derived cardiac progenitors (CPs) or MNPs and leveraging the deep learning approach BPNet, we identified motifs of other TFs that predicted ISL1 occupancy in each lineage, with NKX2.5 and GATA motifs being most closely associated to ISL1 in CPs. Experimentally, nearly two-thirds of ISL1-bound loci were co-occupied by NKX2.5 and/or GATA4. Removal of NKX2.5 from CPs led to widespread ISL1 redistribution, and overexpression of NKX2.5 in MNPs led to ISL1 occupancy of CP-specific loci. These results reveal how ISL1 guides lineage choices through a combinatorial code that dictates genomic occupancy and transcription.
Subject(s)
Induced Pluripotent Stem Cells , Transcription Factors , Humans , Transcription Factors/metabolism , Myocytes, Cardiac , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Induced Pluripotent Stem Cells/metabolism , Cell Differentiation/genetics , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolismABSTRACT
BACKGROUND: Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. METHODS: We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.
Subject(s)
Induced Pluripotent Stem Cells , Transcription Factors , Humans , Mice , Animals , Transcription Factors/genetics , Transcription Factors/metabolism , Homeodomain Proteins/genetics , Gene Regulatory Networks , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Mice, Transgenic , Induced Pluripotent Stem Cells/metabolism , Heart , Myocytes, Cardiac/metabolism , Gene Expression Regulation, DevelopmentalABSTRACT
Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.
Subject(s)
Atrial Fibrillation , Zebrafish , Animals , Gene Expression Regulation, Developmental , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Homeodomain Proteins/metabolism , Myocytes, Cardiac/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The cardiac developmental network has been associated with myocardial regenerative potential. However, the embryonic signals triggered following injury have yet to be fully elucidated. Nkx2.5 is a key causative transcription factor associated with human congenital heart disease and one of the earliest markers of cardiac progenitors, thus it serves as a promising candidate. Here, we show that cardiac-specific RNA-sequencing studies reveal a disrupted embryonic transcriptional profile in the adult Nkx2.5 loss-of-function myocardium. nkx2.5-/- fish exhibit an impaired ability to recover following ventricular apex amputation with diminished dedifferentiation and proliferation. Complex network analyses illuminate that Nkx2.5 is required to provoke proteolytic pathways necessary for sarcomere disassembly and to mount a proliferative response for cardiomyocyte renewal. Moreover, Nkx2.5 targets embedded in these distinct gene regulatory modules coordinate appropriate, multi-faceted injury responses. Altogether, our findings support a previously unrecognized, Nkx2.5-dependent regenerative circuit that invokes myocardial cell cycle re-entry, proteolysis, and mitochondrial metabolism to ensure effective regeneration in the teleost heart.
Subject(s)
Myocardium , Myocytes, Cardiac , Animals , Heart Ventricles/metabolism , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Transcription Factors/metabolismABSTRACT
Heart organoids have the potential to generate primary heart-like anatomical structures and hold great promise as in vitro models for cardiac disease. However, their properties have not yet been fully studied, which hinders their wide spread application. Here we report the development of differentiation systems for ventricular and atrial heart organoids, enabling the study of heart diseases with chamber defects. We show that our systems generate chamber-specific organoids comprising of the major cardiac cell types, and we use single cell RNA sequencing together with sample multiplexing to characterize the cells we generate. To that end, we developed a machine learning label transfer approach leveraging cell type, chamber, and laterality annotations available for primary human fetal heart cells. We then used this model to analyze organoid cells from an isogeneic line carrying an Ebstein's anomaly associated genetic variant in NKX2-5, and we successfully recapitulated the disease's atrialized ventricular defects. In summary, we have established a workflow integrating heart organoids and computational analysis to model heart development in normal and disease states.
Subject(s)
Induced Pluripotent Stem Cells , Organoids , Heart , Heart Ventricles , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Humans , Organogenesis/genetics , Organoids/metabolismABSTRACT
Genome-wide association studies (GWAS) for atrial fibrillation (AF) have uncovered numerous disease-associated variants. Their underlying molecular mechanisms, especially consequences for mRNA and protein expression remain largely elusive. Thus, refined multi-omics approaches are needed for deciphering the underlying molecular networks. Here, we integrate genomics, transcriptomics, and proteomics of human atrial tissue in a cross-sectional study to identify widespread effects of genetic variants on both transcript (cis-eQTL) and protein (cis-pQTL) abundance. We further establish a novel targeted trans-QTL approach based on polygenic risk scores to determine candidates for AF core genes. Using this approach, we identify two trans-eQTLs and five trans-pQTLs for AF GWAS hits, and elucidate the role of the transcription factor NKX2-5 as a link between the GWAS SNP rs9481842 and AF. Altogether, we present an integrative multi-omics method to uncover trans-acting networks in small datasets and provide a rich resource of atrial tissue-specific regulatory variants for transcript and protein levels for cardiovascular disease gene prioritization.
Subject(s)
Atrial Fibrillation/genetics , Genomics , Organ Specificity , Gene Expression Regulation , Genetic Predisposition to Disease , Genome-Wide Association Study , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Humans , Polymorphism, Single Nucleotide/genetics , Quantitative Trait Loci/geneticsABSTRACT
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.
Subject(s)
DEAD-box RNA Helicases , G-Quadruplexes , Myocytes, Cardiac , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Cardiomyopathies/pathology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA/metabolism , Heart Ventricles , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Protein Processing, Post-Translational , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Changes in DNA methylation are associated with normal cardiogenesis, whereas altered methylation patterns can occur in congenital heart disease. Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA demethylation. Here, we characterize stage-specific methylation dynamics and the function of TETs during human cardiomyocyte differentiation. Human embryonic stem cells (hESCs) in which all three TET genes are inactivated fail to generate cardiomyocytes (CMs), with altered mesoderm patterning and defective cardiac progenitor specification. Genome-wide methylation analysis shows TET knockout causes promoter hypermethylation of genes encoding WNT inhibitors, leading to hyperactivated WNT signaling and defects in cardiac mesoderm patterning. TET activity is also needed to maintain hypomethylated status and expression of NKX2-5 for subsequent cardiac progenitor specification. Finally, loss of TETs causes a set of cardiac structural genes to fail to be demethylated at the cardiac progenitor stage. Our data demonstrate key roles for TET proteins in controlling methylation dynamics at sequential steps during human cardiac development.
Subject(s)
Cell Differentiation , DNA Methylation , DNA-Binding Proteins/metabolism , Dioxygenases/metabolism , Epigenesis, Genetic , Human Embryonic Stem Cells/enzymology , Mixed Function Oxygenases/metabolism , Myocytes, Cardiac/enzymology , Proto-Oncogene Proteins/metabolism , DNA-Binding Proteins/genetics , Dioxygenases/genetics , Gene Expression Regulation, Neoplastic , HEK293 Cells , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mixed Function Oxygenases/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Troponin I/genetics , Troponin I/metabolism , Wnt Signaling Pathway/geneticsABSTRACT
The therapeutic efficacy of radiofrequency ablation (RFA) against liver cancer is often limited by proliferation and metastasis of residual tumor cells. These phenomena are closely associated with the Warburg effect, wherein ErbB2 is activated. While RFA inhibits the Warburg effect of residual tumor cells at the early stage, the specific mechanisms remain unclear. We explored the regulatory relationship between the long noncoding RNA ENST00000570843.1 (lncENST) and ErbB2 using lentiviral transfection of lncENST and ErbB2 overexpression/interference vectors in in vitro and in vivo models of hepatocellular carcinoma in the presence of sublethal heat at 50°C. ErbB2-mediated Warburg effect was suppressed by lncENST, as manifested by reduced glucose uptake and lactic acid production in SMMC-7721 cells. lncENST also increased tumor apoptosis and inhibited tumor progression in nude Balb/c mice for up to 28 days after RFA. Additionally, we predicted through bioinformatic analysis that the promoter of ErbB2 binds to the transcription factor Nkx2-5, resulting in a negative regulatory effect. This speculation was confirmed by chromatin immunoprecipitation of the Nkx2-5 protein and ErbB2, indicating that ErbB2 transcription was curbed by Nkx2-5. We propose that lncENST downplays the Warburg effect in residual tumor cells by downregulating ErbB2 via Nkx2-5 activation. This study is aimed at providing molecular targets that can prevent residual tumor cell proliferation after RFA, with clinical significance in hepatocellular carcinoma treatment.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Homeobox Protein Nkx-2.5/metabolism , RNA, Long Noncoding/genetics , Receptor, ErbB-2/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Computational Biology , Disease Progression , Female , Heterografts , Humans , Liver Neoplasms , Mice , Mice, Inbred BALB C , Mice, Nude , Radiofrequency Ablation , Receptor, ErbB-2/genetics , Warburg Effect, OncologicABSTRACT
The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.
Subject(s)
Cell Differentiation/drug effects , Gene Expression Regulation, Developmental/drug effects , Morpholines/toxicity , Mouse Embryonic Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Piperidines/toxicity , Toxicity Tests , Animals , Cells, Cultured , Gene Regulatory Networks , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Mice , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Retinoic Acid 4-Hydroxylase/genetics , Retinoic Acid 4-Hydroxylase/metabolism , Risk Assessment , Spiro Compounds/toxicity , Time Factors , Tubulin/genetics , Tubulin/metabolismABSTRACT
The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.