Early Human Heart Research Articles

Abstract P3101: Chromatin Remodeling BAF (SWI/SNF) Complex Controls Both Cardiogenesis And Neurogenesis From Human Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. Results: Here, we report ARID1A, a DNA-binding subunit of the BAF(SWI/SNF) epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Loss-of-ARID1A (ARID1A –/– ) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A –/– hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF. Conclusions: We uncover the key and opposite roles by the BAF(SWI/SNF) complex to govern early cardiac and neural development. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A-REST/NRSF interaction.

Sequencing of a Chinese tetralogy of Fallot cohort reveals clustering mutations in myogenic heart progenitors.

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.

Abstract MP142: Genome-wide Studies Reveal the Essential and Opposite Roles of Arid1a in Controlling Human Cardiogenesis and Neurogenesis From Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene/complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. The evolutionarily conserved ATP-dependent SWI/SNF complex is one of the largest chromatin remodeling complexes, consisting of ~15 subunits, including SMARCA2 (also known as BRM) or SMARCA4 (also known as BRG1) as the ATPase catalytic subunit. Several BRG1-associated factors (BAFs), such as ARID1A (Baf250a), have DNA binding capacity and assemble with either BRM or BRG1 to form a functional chromatin-remodeling complex. A single amino acid mutation (Arid1aV 1068G/V1068G ), impaired Arid1a-DNA interactions and resulted in both cardiac neural defects. Mutations in 4 different SWI/SNF subunits including ARID1A/B were identified in human congenital syndromes that include both neural and cardiac defects. Results: Here, we report ARID1A, which is a DNA-binding-subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) via employing distinct mechanisms. CRISPR/Cas-9 knockout of ARID1A (ARID1A -/- ) led to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A -/- hESCs was prominently suppressed, whereas neural differentiation was significantly promoted. Whole genome-wide ChIP-seq and ATAC-seq analyses revealed that ARID1A was required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during neural development, transcription of most essential neurogenic genes was dependent on ARID1A and ARID1A could interact with REST, which is a known transcriptional repressor. Conclusions: We uncovered the key and opposite roles by ARID1A to govern both early human cardiac and neural development and characterized the mechanisms. We found global chromatin accessibility on cardiogenic genes was dependent on ARID1A, whereas transcriptional activity of neurogenic genes was regulated by ARID1A, possibly through ARID1A-REST interaction.

Genome-wide studies reveal the essential and opposite roles of ARID1A in controlling human cardiogenesis and neurogenesis from pluripotent stem cells

BackgroundEarly human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive.ResultsHere, we report ARID1A, a DNA-binding subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Knockout-of-ARID1A (ARID1A−/−) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A −/− hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF.ConclusionsWe uncover the opposite roles by ARID1A to govern both early cardiac and neural development from pluripotent stem cells. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A, possibly through ARID1A-REST/NRSF interaction.

Multiple roles and regulatory mechanisms of the transcription factor GATA6 in human cancers.

Cancer is a common type of non-communicable disease, and its morbidity and mortality are rapidly increasing. It is expected to become the largest obstacle to the promotion of global human health in the future. Some transcription factors that play important regulatory roles in embryogenesis and subsequent tissue maintenance can be selectively amplified during tumorigenesis. Due to its high expression in the embryonic endoderm and mesoderm, GATA6 plays a crucial role in the normal development of early human heart, lung, digestive system, adrenal glands, breasts, ovaries, retina, skin, and nervous system. Up to now, overexpression of the GATA6 gene has been shown to play an important role in several cancers, including lung cancer, digestive system tumors, breast cancer, and ovarian cancer. However, the human body is a complex organism, which causes the transcription factor GATA6 to have multiple roles in cancer. In this review, we summarize the multiple roles of transcription factor GATA6 in various cancers and its regulatory mechanisms. The aim is to better understand the relationship between GATA6 gene expression and cancer development and to provide new insights for exploring potential therapeutic targets.

Prenatal hypoxia programs adult cardiac contractile dysfunction through changes in beta‐adrenergic signaling

Prenatal hypoxia leads to fetal growth restriction and increased risk of adult cardiovascular disease. We have previously shown that adult cardiac beta‐adrenergic receptor (βAR) desensitization is programmed by prenatal hypoxia. Our objective was to determine how the observed βAR desensitization affects cardiac contractility in vivo and whether it is linked to changes in cAMP accumulation, β1AR/β2AR ratio or G‐protein expression. Eggs were incubated in normoxia (N, 20.95% O2) or hypoxia from day 0 (H, 14% O2), hatched and raised to 5wks of age. Echocardiograms were recorded in anesthetized birds before and after isoproterenol stimulation. cAMP accumulation in isolated cells after stimulation with isoproterenol, β1AR/β2AR ratio and Gs and Gi expression in tissue was also assessed. Prenatal hypoxia caused contractile dysfunction as shown by a decrease in systolic diameter. cAMP levels after βAR stimulation and β1AR/β2AR ratio were lower in hypoxic incubated birds. Gi expression was unchanged while Gs was increased. Our results resemble those reported in early human heart failure. We conclude that prenatal hypoxia has a programming effect on adult βAR function and speculate that the 5 week chicken exposed to prenatal hypoxia is displaying early signs of heart failure. Supported by FORMAS Centre of Excellence in Animal Welfare Science and career grant from Linköpings universitet to JA.

Formation of the Building Plan of the Human Heart

The intricate process of formation of the heart relies on the spatiotemporal regulation of differentiation and growth of its different parts. Errors in these processes can result in hypoplasia of the chambers and incorrect alignment of the atria and great arteries with the ventricles, with such abnormalities producing the worst forms of congenital heart disease.1 Although recent advances in molecular embryology have greatly improved our understanding of normal cardiac development, our insights into the pathogenesis of human cardiac malformations remain limited.2 With the current lack of data on gene expression in humans, inferences concerning morphogenesis rely on classic studies of human cardiac development or on extrapolation of experimental data from animal studies. Most textbooks on human embryology describe the embryonic heart tube as already possessing a linear array of the definitive components, despite this so-called segmental concept being disproved some time ago by fate-mapping experiments in chickens,3,4 which are now endorsed by recent molecular lineage analyses in mice.5 Proliferation studies in chickens revealed that the primary heart tube proliferates minimally and that its growth occurs through addition of differentiating cells from visceral mesoderm at the venous and arterial poles.6 These findings underscore previous lineage studies performed in mice, which have shown that transcription factors Islet1 and Tbx1 play a crucial role in the intricate balance between the cardiac precursor state and differentiation.7,8 Subsequent to its formation, the heart tube elongates and loops, producing its inner and outer curvatures and providing the general building plan for formation of the chambers and conduction system.9 The chambers themselves form by local proliferation and differentiation at the outer curvature.10,11 At the same time, the differentiating chamber myocardium remains flanked by primary myocardium, which is prevented from further differentiation by the …

Neuregulin/ErbB Signaling Regulates Cardiac Subtype Specification in Differentiating Human Embryonic Stem Cells

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes. To demonstrate intact neuregulin (NRG)-1β/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1β, an anti-NRG-1β neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1β/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. NRG-1β/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1β/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker.

Quantitative Proteomics Profiling of Sarcomere Associated Proteins in Limb and Extraocular Muscle Allotypes

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: Implication for the analysis of early human heart disease

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.

The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart.

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease. (Mol Cell Biochem 263: 91-97, 2004).