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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.

NKX2-5, a master cardiac regulatory transcription factor was the first known genetic cause of congenital heart diseases (CHDs). To further investigate its role in CHD pathogenesis, we performed mutational screening of 285 CHD probands and 200 healthy controls. Five coding sequence variants were identified in six CHD cases (2.1%), including three in the N-terminal region (p.A61G, p.R95L, and p.E131K) and one each in homeodomain (HD) (p.A148E) and tyrosine-rich domain (p.P247A). Variant-p.A148E showed tertiary structure changes and differential DNA binding affinity of mutant compared to wild type. Two N-terminal variants-p.A61G and p.E131K along with HD variant p.A148E demonstrated significantly reduced transcriptional activity of Nppa and Actc1 promoters in dual luciferase promoter assay supported by their reduced expression in qRT-PCR. Nonetheless, variant p.R95L affected the synergy of NKX2-5 with serum response factor and TBX5 leading to significantly decreased Actc1 promoter activity depicting a distinctive role of this region. The aberrant expression of other target genes-Irx4, Mef2c, Bmp10, Myh6, Myh7, and Myocd is also observed in response to NKX2-5 variants, possibly due to the defective gene regulatory network. Severely impaired downstream promoter activities and abnormal expression of target genes due to N-terminal variants supports the emerging role of this region during cardiac-developmental pathways.

It has been shown that near all organs, especially the cardiovascular system, are affected by bacterial lipopolysaccharide via the activation of Toll-like receptor signaling pathways. Here, we tried to find the blunting effect of bacterial lipase on lipopolysaccharide (LPS)-induced cardiac tissue toxicity in chicken embryos. 7-day fertilized chicken eggs were divided randomly into different groups as follows; Control, Normal Saline, LPS (0.1, 0.5 and 1 mg/kbw), and LPS (0.1, 0.5 and 1 mg/kbw) plus 5 mg/ml Lipase. On day 17, the hearts were sampled. The expression of genes such as GATA4, NKX2.5, EGFR, TRIF, and NF-ƙB was monitored using real-time PCR analysis. Using western blotting, we measured NF-ƙB protein level. Total antioxidant capacity, glutathione peroxidase, and Catalase activity were also studied. Microvascular density and anterior wall thickness were monitored in histological samples using HE staining. High dose of LPS (1 mg/kbw) increased the expression of TRIF but not NF-ƙB compared to the control group (p 0.05). We found a statistically significant reduction in groups that received LPS + Lipase compared to the control and LPS groups (p 0.05). Western blotting revealed that the injection of Lipase could reduce LPS-induced NF-ƙB compared to the control group (p 0.05). The expression of GATA4, NKx2.5, and EGFR was not altered in the LPS group, while the simultaneous application of LPS and Lipase significantly reduced GATA4, NKx2.5, and EGFR levels below the control (p 0.05). We found non-significant differences in glutathione peroxidase, and Catalase activity in all groups (p 0.05), while total antioxidant capacity was increased in groups that received LPS + Lipase. Anterior wall thickness was diminished in LPS groups and the use of both lipase and LPS returned near-to-control values (p 0.05). Despite a slight increase in microvascular density, we found statistically non-significant differences in all groups (p 0.05). Bacterial lipase reduces detrimental effects of LPS on chicken embryo heart induced via Toll-like receptor signaling pathway.

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers (PDGFR-α, FLK1, ISL1, NKX2.5, CTNT, CX43 and MYHC-B) in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22 and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSC). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and human iPSC, with an earlier onset in the hiPSC compared to reported murine expression. Markers of immature cardiomyocytes, CTNT and MYHC-B were consistently expressed throughout E16 - E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, whilst this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm, ISL1/NKX2.5 for cardiac progenitors and CTNT/MYHC-B for immature cardiomyocytes. Further, CTNT+/ISL1+ could mark immature cardiomyocytes and CTNT+/ISL1- mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo like cardiac tissue in the future.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice., Heart-forming organoids model early cardiac development.

Bmp plays an important role in cardiomyocyte differentiation, but the function of Smad4 in Bmp signaling remains elusive. Here, we show that disruption of the Smad4 gene in cardiac progenitors expressing Sfrp5 led to embryonic lethality with hypoplastic heart formation. Although the expression of Nkx2-5 is regulated by Bmp signaling, expression of Nkx2-5 was weakly detected in the mutant heart. However, the nuclear translocation of Nkx2-5 was impaired. Expression of CK2 or PP1, which could alter the phosphorylation status of the NLS of Nkx2-5, was not affected, but Nkx2-5 was found to bind to Smad4 by co-immunoprecipitation experiments. Introduction of Smad4 into cells derived from Smad4 conditional knockout embryonic hearts restored the nuclear localization of Nkx2-5, and exogenous Nkx2-5 failed to translocate into the nucleus of Smad4-depleted fibroblasts. These results suggest that Smad4 plays an essential role in cardiomyocyte differentiation by controlling not only transcription but also the nuclear localization of Nkx2-5.

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.

Mutations of NKX2-5 largely contribute to congenital heart diseases (CHDs), especially atrial septal defect (ASD). We identified a novel heterozygous splicing mutation c.335-1G gt; A in NKX2-5 gene in an ASD family via whole exome sequencing (WES) and linkage analysis. Utilizing the human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a disease model, we showed that haploinsufficiency of NKX2-5 contributed to aberrant orchestration of apoptosis and proliferation in ASD patient-derived hiPSC-CMs. RNA-seq profiling and dual-luciferase reporter assay revealed that NKX2-5 acts upstream of PYK2 via miR-19a and miR-19b (miR-19a/b) to regulate cardiomyocyte apoptosis. Meanwhile, miR-19a/b are also downstream mediators of NKX2-5 during cardiomyocyte proliferation. The novel splicing mutation c.335-1G gt; A in NKX2-5 and its potential pathogenic roles in ASD were demonstrated. Our work provides clues not only for deep understanding of NKX2-5 in cardia development, but also for better knowledge in the molecular mechanisms of CHDs.

(1) Background globe. The etiology of CHDs is complex and involves both genetic and non-genetic factors. Although, significant progress has been made in deciphering the genetic components involved in CHDs, recent reports have revealed that mutations in Nk2 homeobox5 (

BVES is a transmembrane protein, our previous work demonstrated that single nucleotide mutations of BVES in tetralogy of fallot (TOF) patients cause a downregulation of BVES transcription. However, the relationship between BVES and the pathogenesis of TOF has not been determined. Here we reported our research results about the relationship between BVES and the right ventricular outflow tract (RVOT) stenosis. BVES expression was significantly downregulated in most TOF samples compared with controls. The expression of the second heart field (SHF) regulatory network genes, including NKX2.5, GATA4 and HAND2, was also decreased in the TOF samples. In zebrafish, bves knockdown resulted in looping defects and ventricular outflow tract (VOT) stenosis, which was mostly rescued by injecting bves mRNA. bves knockdown in zebrafish also decreased the expression of SHF genes, such as nkx2.5, gata4 and hand2, consistent with the TOF samples` results. The dual-fluorescence reporter system analysis showed that BVES positively regulated the transcriptional activity of GATA4, NKX2.5 and HAND2 promoters. In zebrafish, nkx2.5 mRNA partially rescued VOT stenosis caused by bves knockdown. These results indicate that BVES downregulation may be associated with RVOT stenosis of non-syndromic TOF, and bves is probably involved in the development of VOT in zebrafish.

Increased application of the pyrethroid insecticide deltamethrin has adverse effects on the cardiac system and neurobehavior on the non-target organisms, which has raised the public's attention. Because of spermidine and melatonin considered to have cardioprotective and neuroprotective characteristics, zebrafish were utilized as the model organism to explore the protective effects of spermidine and melatonin against deltamethrin-induced toxicity. We tested the neurobehavior of zebrafish larvae through a rest/wake behavior assay, and evaluated the levels of the expression of Scn5lab, gata4, nkx2.5, hcrt, hcrtr, and aanat2 by qRT-PCR. Besides that cmlc2 was evaluated by whole-mount in situ hybridization. Results have shown that compared with control group, 0.025 mg/L deltamethrin could significantly disturb the cardiac development, downregulating the expression of Scn5lab and transcriptional factors gata4 and nkx2.5, disturbing cardiac looping, resulting in defects in cardiac morphology and function. Moreover, deltamethrin could alter the expression levels of rest/wake genes and cause hyperactivity in zebrafish larvae. Besides, compared with deltamethrin group, the exogenous 0.01 mg/L spermidine and 0.232 mg/L melatonin could significantly rescue the adverse effects of deltamethrin on the cardiac system and neurobehavior in zebrafish. This indicated that spermidine and melatonin have neuroprotective and cardioprotective effects against deltamethrin-induced adverse effects in zebrafish.

NKX2-5 is a homeodomain transcription factor that plays a crucial role in heart development. It is the first gene where a single genetic variant (GV) was found to be associated with congenital heart diseases in humans. In this study, we carried out a comprehensive survey of NKX2-5 GVs to build a unified, curated, and updated compilation of all available GVs. We retrieved a total of 1,380 unique GVs. From these, 970 had information on their frequency in the general population and 143 have been linked to pathogenic phenotypes in humans. In vitro effect was ascertained for 38 GVs. The homeodomain had the biggest cluster of pathogenic variants in the protein: 49 GVs in 60 residues, 23 in its third α-helix, where 11 missense variants may affect protein–DNA interaction or the hydrophobic core. We also pinpointed the likely location of pathogenic GVs in four linear motifs. These analyses allowed us to assign a putative explanation for the effect of 90 GVs. This study pointed to reliable pathogenicity for GVs in helix 3 of the homeodomain and may broaden the scope of functional and structural studies that can be done to better understand the effect of GVs in NKX2-5 function. Fil: Kolomenski, Jorge Emilio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Delea, Marisol. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; Argentina Fil: Simonetti, Leandro. Uppsala Universitet; Suecia Fil: Fabbro, Mónica C.. No especifíca; Fil: Espeche, Lucia Daniela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; Argentina Fil: Taboas, Melisa. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; Argentina Fil: Nadra, Alejandro Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bruque, Carlos David. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Dain, Liliana Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas)., Development, 149 (3), ISSN:0950-1991, ISSN:1477-9129

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., Numerous disease-associated variants have been described in GWAS for atrial fibrillation. Here the authors integrate omics data to investigate the consequences of genetic variants for transcript and protein levels in the atrium of the human heart. With this multi-omics approach, authors reveal the regulatory network underlying atrial fibrillation and provide a resource for cardiac gene prioritization.

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.

Background NKX2-5 variant in atrial septal defect patients has been reported. However, it is not yet been described in the Southeast Asian population. Here, we screened the NKX2-5 variants in patients with atrial septal defect (ASD) in the Indonesian population. Method We recruited 97 patients with ASD for genetic screening of the NKX2-5 variant using Sanger sequencing. Results We identified three variants of NKX2-5: NM_004387.4:c.63A>G at exon 1, NM_004387.4:c.413G>A, and NM_004387.4:c.561G>C at exon 2. The first variant is commonly found (85.6%) and benign. The last two variants are heterozygous at the same locus. These variants are rare (3.1%) and novel. Interestingly, these variants were discovered in familial atrial septal defects with a spectrum of arrhythmia and severe pulmonary hypertension. Conclusion Our study is the first report of the NKX2-5 variant in ASD patients in the Southeast Asian population, including a novel heterozygous variant: NM_004387.4:c.413G>A and NM_004387.4:c.561G>C. These variants might contribute to familial ASD risk with arrhythmia and severe pulmonary hypertension. Functional studies are necessary to prove our findings.

Patent Foramen Ovale (PFO) is a common postnatal defect of cardiac atrial septation. A certain degree of familial aggregation has been reported. Animal studies suggest the involvement of the Notch pathway and other cardiac transcription factors (GATA4, TBX20, NKX2-5) in Foramen Ovale closure. This review evaluates the contribution of genetic alterations in PFO development. We systematically reviewed studies that assessed rare and common variants in subjects with PFO. The protocol was registered with PROSPERO and followed MOOSE guidelines. We systematically searched English studies reporting rates of variants in PFO subjects until the 30th of June 2021. Among 1231 studies, we included four studies: two of them assessed the NKX2-5 gene, the remaining reported variants of chromosome 4q25 and the GATA4 S377G variant, respectively. We did not find any variant associated with PFO, except for the rs2200733 variant of chromosome 4q25 in atrial fibrillation patients. Despite the scarceness of evidence so far, animal studies and other studies that did not fulfil the criteria to be included in the review indicate a robust genetic background in PFO. More research is needed on the genetic determinants of PFO.

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.

MicroRNAs (miRNAs) are translational regulatory molecules with recognised roles in heart development and disease. Therefore, it is important to define the human miRNA expression profile in cardiac progenitors and early-differentiated cardiomyocytes and to determine whether critical cardiac transcription factors such as NKX2-5 regulate miRNA expression. We used an NKX2-5eGFP/w reporter line to isolate both cardiac committed mesoderm and cardiomyocytes. We identified 11 miRNAs that were differentially expressed in NKX2-5 -expressing cardiac mesoderm compared to non-cardiac mesoderm. Subsequent profiling revealed that the canonical myogenic miRNAs including MIR1-1, MIR133A1 and MIR208A were enriched in cardiomyocytes. Strikingly, deletion of NKX2-5 did not result in gross changes in the cardiac miRNA profile, either at committed mesoderm or cardiomyocyte stages. Thus, in early human cardiomyocyte commitment and differentiation, the cardiac myogenic miRNA program is predominantly regulated independently of the highly conserved NKX2-5 -dependant gene regulatory network.

Background: Tetralogy of Fallot (TOF) accounts for ∼10% of congenital heart disease cases. The blood vessel epicardial substance (BVES) gene has been reported to play a role in the function of adult hearts. However, whether allelic variants in BVES contribute to the risk of TOF and its possible mechanism remains unknown. Methods: The open reading frame of the BVES gene was sequenced using samples from 146 TOF patients and 100 unrelated healthy controls. qRT-PCR and western blot assays were used to confirm the expression of mutated BVES variants in the TOF samples. The online software Polyphen2 and SIFT were used to predict the deleterious effects of the observed allelic variants. The effects of these allelic variants on the transcriptional activities of genes were examined using dual-fluorescence reporter assays. Results: We genotyped four single nucleotide polymorphisms (SNPs) in the BVES gene from each of the 146 TOF patients. Among them, the minor allelic frequencies of c.385C>T (p.R129W) were 0.035% in TOF, but ∼0.025% in 100 controls and the Chinese Millionome Database. This allelic variant was predicted to be a potentially harmful alteration by the Polyphen2 and SIFT softwares. qRT-PCR and western blot analyses indicated that the expression of BVES in the six right ventricular outflow tract samples with the c.385C>T allelic variant was significantly downregulated. A dual-fluorescence reporter system showed that the c.385C>T allelic variant significantly decreased the transcriptional activity of the BVES gene and also decreased transcription from the GATA4 and NKX2.5 promoters. Conclusions: c.385C>T (p.R129W) is a functional SNP of the BVES gene that reduces the transcriptional activity of BVES in vitro and in vivo in TOF tissues. This subsequently affects the transcriptional activities of GATA4 and NKX2.5 related to TOF. These findings suggest that c.385C>T may be associated with the risk of TOF in the Han Chinese population.

Rationale: We hypothesized that the differentiation processes of cardiac progenitor cell (CP) from first and second heart fields (FHF and SHF) may undergo the unique instructive gene regulatory networks or signaling pathways, and the precise SHF progression is contingent on the FHF signaling developmental cues. Objective: We investigated how the intraorgan communications control sequential building of discrete anatomic regions of the heart at single-cell resolution. Methods and Results: By single-cell transcriptomic analysis of Nkx2-5 (NK2 homeobox 5) and Isl1 (ISL LIM homeobox 1) lineages at embryonic day 7.75, embryonic day 8.25, embryonic day 8.75, and embryonic day 9.25, we present a panoramic view of distinct CP differentiation hierarchies. Computational identifications of FHF- and SHF-CP descendants revealed that SHF differentiation toward cardiomyocytes underwent numerous step-like transitions, whereas earlier FHF progressed toward cardiomyocytes in a wave-like manner. Importantly, single-cell pairing analysis demonstrated that SHF-CPs were attracted to and expanded FHF-populated heart tube region through interlineage communications mediated by the chemotactic guidance (MIF [macrophage migration inhibitory factor]-CXCR2 [C-X-C motif chemokine receptor 2]). This finding was verified by pharmacological blockade of this chemotaxis in embryos manifesting limited SHF cell migration and contribution to the growth of the outflow tract and right ventricle but undetectable effects on the left ventricle or heart tube initiation. Genetic loss-of-function assay of Cxcr2 showed that the expression domain of CXCR4 was expanded predominantly at SHF. Furthermore, double knockout of Cxcr2/Cxcr4 exhibited defective SHF development, corroborating the redundant function. Mechanistically, NKX2-5 directly bound the Cxcr2 and Cxcr4 genomic loci and activated their transcription in SHF. Conclusions: Collectively, we propose a model in which the chemotaxis-mediated intraorgan crosstalk spatiotemporally guides the successive process of positioning SHF-CP and promoting primary heart expansion and patterning upon FHF-derived heart tube initiation.

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958–972

Three rights can make a wrong Many diseases are thought to arise from co-inheritance of rare genetic variants that are benign on their own but harmful in combination. This hypothesis has been difficult to validate by functional experiments. Gifford et al. sequenced the genomes of two parents who were asymptomatic and their three children, all of whom had early-onset heart disease. They identified three likely culprit genetic variants, two in transcription factor genes linked to heart development and one in a gene encoding a muscle structural protein. When they introduced these three variants together into mice by gene editing, the mice developed heart disease resembling that seen in the children. Science , this issue p. 865

BACKGROUND NOTCH1 plays a key role in the differentiation of ventricles, and mutations are strongly associated with both sporadic and familial bicuspid aortic valves. However, few heart-specific enhancers have been identified to date. RESULTS In this study, we investigated evolutionary conserved regions (ECRs) that might act as potential enhancers within the region approximately 150-kb upstream and downstream of the NOTCH1 gene. Functional validation revealed that one 127-bp ECR located ~85-kb downstream of the NOTCH1 gene drives green fluorescent protein expression in the zebrafish embryo heart. Transcription factor (TF) prediction and core TF distribution analyses were performed to identify the core region. Dissection of ECR3 was performed to identify the 42-bp sequence, which is sufficient for heart-specific expression. In situ hybridization experiments showed that notch1b is expressed in the heart. Overexpression experiments in cells indicated that NKX2-5 is critical for enhancer activity. Mutation of the NKX-5 binding site significantly decreased reporter gene expression. Next, compared with the commonly used myocardium-labeled zebrafish transgenic strain Tg(cmlc2: mCherry), this 42-bp enhancer-labeled stable line mediated a similar expression pattern but with a smaller core region. CONCLUSION This study identified a 42-bp heart-specific enhancer near the NOTCH1 gene and further verified its functional targeting by NKX2-5.

We systematically investigate the applicability of a molecular dynamics-based setup for the calculations of standard binding free energies of biologically relevant protein-DNA complexes. The free energies are extracted from a potential of mean force calculated using umbrella sampling simulations. Two protein-DNA systems derived from a homeodomain transcription factor complex are studied in order to investigate the binding of both disordered and globular proteins. Free energies and trajectories obtained using two modern molecular mechanical force fields are compared to each other and to experimental data. The temperature dependence of the calculated standard binding free energies is investigated by performing all simulations over a range of temperatures. We show that the values of standard binding free energies obtained from these simulations are overestimated compared to experimental results. Significant differences are observed between the two protein-DNA systems and between the two force fields, which are explained by different propensities to form inter- and intramolecular contacts. The number of protein-DNA contacts increases with increasing temperature, in agreement with the experimentally known temperature dependence of enthalpies of binding. However, conclusions about the temperature dependence of the standard binding free energies cannot be made with confidence, as the differences among the values are on the order of statistical uncertainty.

Cardiac differentiation in vitro is a complex, stepwise process that is rigidly governed by a subset of transcription factors and signaling cascades. In this study, we investigated the cooperation of cardiac-specific transcription factors Gata4 and Nkx2.5, as well as mitogen-activated protein kinase (MAPK) cascades. P19 embryonic carcinoma cells were induced into spontaneously beating cardiomyocytes utilizing a two-step protocol that comprised an early stage and a late stage of differentiation. During early-stage differentiation in suspension culture, P19 cells aggregated to form embryoid bodies (EBs), and the Gata4 and Nkx2.5 genes were induced. However, Gata4 expressed at the early stage of differentiation was incapable of activating downstream gene expression, as it was localized in the cytoplasm and prone to degradation. After EBs were plated for late-stage differentiation in adherent culture, the MAPK cascades were highly activated and contributed to the activation of Gata4 and Nkx2.5. Specifically, we revealed that p38 signaling participated in regulating the localization and stabilization of Gata4 and Nkx2.5. Additionally, the JNK cascade regulated late-stage cardiac differentiation; JNK kinase reduced Gata4 stabilization and conversely alleviated Nkx2.5 degradation by direct interaction and phosphorylation of Nkx2.5. Finally, we found that the C-terminal domain of Nkx2.5 was required for its stabilization under conditions of oxidative stress and JNK activation. Overall, our results indicated that the induction and activation of Gata4 and Nkx2.5 during early- and late-stage cardiac differentiation was closely associated with the function of the MAPK signaling cascades.

Heart failure is a heterogeneous disease with multiple risk factors and various pathophysiological types, which makes it difficult to understand the molecular mechanisms involved. In this study, we proposed a trans-omics approach for predicting molecular pathological mechanisms of heart failure and identifying marker genes to distinguish heterogeneous phenotypes, by integrating multiple omics data including single-cell RNA-seq, ChIP-seq, and gene interactome data. We detected a significant increase in the expression level of natriuretic peptide A (Nppa), after stress loading with transverse aortic constriction (TAC), and showed that cardiomyocytes with high Nppa expression displayed specific gene expression patterns. Multiple NADH ubiquinone complex family, which are associated with the mitochondrial electron transport system, were negatively correlated with Nppa expression during the early stages of cardiac hypertrophy. Large-scale ChIP-seq data analysis showed that Nkx2-5 and Gtf2b were transcription factors characteristic of high-Nppa-expressing cardiomyocytes. Nppa expression levels may, therefore, represent a useful diagnostic marker for heart failure.

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.