Congenital heart defects (CHD) are the most common form of developmental abnormalities, occurring in ~1% of live births, and can arise due to altered dosage of genes essential for cardiogenesis. Aneuploidy is a common cause of CHD, accounting for nearly 15% of cases, and the most common aneuploidy involves trisomy of chromosome 21 (Ch21), resulting in Down Syndrome (DS). CHD is present in ~50% of DS cases, with a 1000-fold enrichment of complete atrioventricular canal defects affecting the junction of the atria and ventricles. This junction establishes valvuloseptal boundaries and is marked by unique myocardial cells distinct from neighboring chamber myocardium. While the presence of a third copy of one or more genes on Ch21 underlies atrioventricular canal and other cardiac defects, the dosage-sensitive CHD gene(s) on Ch21 causing this are not fully defined. Here, using human pluripotent stem cell and mouse models of DS, we identify HMGN1, a nucleosome-binding epigenetic factor on Ch21, as a dosage sensitive regulator of cardiac defects in DS. Single cell transcriptomics revealed that human atrioventricular cardiomyocytes shifted to a more ventricular myocardial state in trisomy 21. CRISPR-activation in disomic cells of 66 candidate Ch21 genes expressed in heart development and single cell RNA-sequencing (CROP-seq) combined with machine learning predictions indicated that increase in HMGN1 altered the transcriptional state of atrioventricular cardiomyocytes similar to trisomy 21. Increased dosage of HMGN1 in cardiomyocytes was associated with altered levels of active chromatin marked by H3K27ac in specific subsets of regulatory regions governing cardiomyocyte maturation. Reduction of Hmgn1 from three to two alleles in a mouse model of DS partially attenuated developmental lethality and incidence of cardiac septal defects. Our results highlight the importance of dosage regulation of epigenetic factors in cardiac development and present a generalizable approach for identifying candidate genes within areas of aneuploidy.

Down syndrome (DS) is caused by a trisomy of human chromosome 21 (Hsa21) and results in a broad range of phenotypes including cognitive impairment, early onset Alzheimer's disease and congenital heart defects (CHD). DS is a gene dosage disorder, with phenotypes arising from an extra copy of one or more of the ~230 genes on Hsa21. However, the genes that are required in three copies to cause congenital heart defects and the underlying mechanisms are unknown. We have generated mouse model for DS (Dp1Tyb) carrying a duplication of 23Mb on Mmu16, orthologous to Hsa21. These mice show CHD such as ventricular septal defects and atrioventricular septal defects which are similar to the CHD seen in babaies with DS. Analysis of the transcriptomic changes in Dp1Tyb mouse model and embryonic hearts from human fetuses with DS show a reduced expression of mitochondrial respiration and cell proliferation genes correlating with CHD. Using systematic genetic mapping, we determine that three copies of the Dyrk1a gene, encoding a serine/threonine protein kinase, are required to cause CHD. Reducing Dyrk1a copy number from three to two rescues defects in proliferation and mitochondrial respiration in embryonic cardiomyocytes and rescues septation defects in Dp1Tyb embryonic hearts. Furthermore, treatment of pregnant mice with a DYRK1A inhibitor developed for clinical use partially rescues the proliferation and mitochondira defects in Dp1Tyb embryos. Thus, increased dosage of DYRK1A is required to impair proliferation mitochondrial function and cause CHD in DS.

Correct formation of the cardiac chambers is essential for proper heart function throughout life. Improper development of the atrial chamber can result in atrial septal defects, a common congenital heart defect. Although vertebrate atrial development is regulated by highly conserved Nr2f transcription factors, little is known about the mechanisms regulating their expression. Here, we show a conserved promoter-associated long non-coding RNA (pa-lncRNA) limits Nr2f translation and restricts atrial size during development. In a zebrafish nr2f1a mutant allele with a deletion in the 5’UTR that does not significantly impact transcription, we found nr2f1a translation is lost and the conserved pa-lncRNA nr2f1-as has increased expression, suggesting nr2f1a-as may regulate nr2f1a expression. CRISPR-Cas9 generated transcriptional start site nr2f1a-as mutants showed overall nr2f1a transcript levels were not affected, yet Nr2f1a protein levels were increased, supporting that nr2f1a-as buffers nr2f1a translation. Furthermore, consistent with nr2f1a-as limiting Nr2f1a protein levels, blocking nr2f1a-as results in increased atrial cardiomyocytes. As overall transcript levels did not correlate with Nr2f1a translation, we determined if ribosomal association of nr2f1a and nr2f1a-as transcripts was affected using translational ribosomal affinity purification assay in these mutants. We detected increased association of nr2f1a transcripts with ribosomes in nr2f1a-as mutants, and increased association of nr2f1a-as in the nr2f1a 5’-UTR mutants. To determine if the translational buffering occurs via direct association of the transcripts, we performed a MS2-tagged RNA affinity purification assay. We found that nr2f1a-as and nr2f1a transcripts can associate with each other. Altogether, these data support a mechanism by which conserved Nr2f pa-lncRNAs limit Nr2f translation through interactions that affect their association with ribosomes, thereby regulating atrial development.

Congenital heart defects occur in approximately 1% of newborns in the United States. Despite their prevalence, less than a third of congenital heart defects can be traced to genetic or environmental causes, indicating that a majority of cardiac malformations arise via poorly understood mechanisms. It is now apparent that hemodynamic cues such as intracardiac blood velocity, blood pressure, and wall shear stress (WSS) directly impact cardiac and vascular development. However, due to the small size and poor accessibility of the embryonic heart, quantitatively capturing the spatiotemporal dynamics of blood flow throughout cardiac development is infeasible experimentally. Major questions remain regarding how hemodynamic forces shape normal and pathological cardiac morphogenesis. Thus, we’ve developed an experimental and computational fluid dynamics (CFD) model pipeline to explore how local tissue geometry variations or perturbed hemodynamics impact the patterning of cardiac regions prone to congenital malformations across a cohort of early hearts. Herein, we used light sheet fluorescence microscopy to generate cell-accurate three-dimensional renderings of a cohort of developing chick hearts. We employed a CFD model described by the full incompressible Navier-Stokes equations, implemented using the finite element method, to simulate pulsatile blood flow through the imaged embryonic hearts to determine how alterations in local anatomies impact hemodynamics in subject-specific anatomies. Furthermore, we have begun computationally and experimentally manipulating parameters to directly impact hemodynamics, such as elevating intracardiac blood viscosity in vivo and computationally, to define how changes in hemodynamics reciprocally change cardiac tissue development. Through our initial validation of these approaches, we have shown that there is little convective influence under this flow regime, and that local stenosis alone within an anatomy is not sufficient for predicting WSS dynamics. Therefore, we have developed a robust CFD modeling pipeline that incorporates detailed cardiac geometries to produce precise descriptions of cardiac hemodynamics across a cohort of embryonic hearts. Moving forward, this pipeline can be used to quickly investigate, in high-resolution, the impact of hemodynamic perturbations on a variety of developing cardiac anatomies.

Congenital heart defects (CHDs) are the most common type of birth defect, accounting for >20% of all deaths in the first year of life, yet the etiology of most CHDs remains unknown. One common genetic cause of CHD is 22q11.2 deletion syndrome (22q11.2DS), where ~75% of 22q11.2DS patients present with CHD. SLC25A1, a gene found within the 22q11.2DS critical deletion region, encodes for the mitochondrial citrate exporter which regulates citrate distribution required for regulating oxidative phosphorylation, glycolysis, and cytosolic Acetyl-CoA production. As the developing heart is a dynamic metabolic environment that undergoes several physiological and morphological transitions before birth, we hypothesize that SLC25A1 plays a key role in metabolic processes that are required for cardiac development. In developing a knockout mouse model of SLC25A1, we uncovered perinatal lethality and hearts from knockout embryos displayed a wide array of congenital heart defects. Mitochondrial structure and functional studies revealed that loss of Slc25a1 causes ultrastructural derangements and decreased oxygen consumption. Transcriptomics analyses of metabolism-related genes revealed that Slc25a1 deletion causes widespread alterations in metabolic gene expression in a dosage-dependent manner. Moreso, metabolic modelling predicted that loss of SLC25A1 downregulates oxidative phosphorylation, while upregulating glycolysis. Additionally, we found that loss of SLC25A1 decreases H3K9 acetylation levels globally and at promoter regions of dysregulated metabolic genes. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis and cardiac metabolic maturation and suggests a role in congenital heart disease.