Congenital heart defects (CHDs) are among the most common birth defects, but their etiology has long been mysterious. In recent decades, the development of a variety of experimental models has led to a greater understanding of the molecular basis of CHDs. In this review, we contrast mouse models of CHD, which maintain the anatomical arrangement of the heart, and human cellular models of CHD, which are more likely to capture human-specific biology but lack anatomical structure. We also discuss the recent development of cardiac organoids, which are a promising step toward more anatomically informative human models of CHD.

Dosage-sensitive transcription factors (TFs) underlie altered gene regulation in human developmental disorders, and cell-type specific gene regulation is linked to the reorganization of 3D chromatin during cellular differentiation. Here, we show dose-dependent regulation of chromatin organization by the congenital heart disease (CHD)- linked, lineage-restricted TF TBX5 in human cardiomyocyte differentiation. Genome organization, including compartments, topologically associated domains, and chromatin loops, are sensitive to reduced TBX5 dosage in a human model of CHD, with variations in response across individual cells. Regions normally bound by TBX5 are especially sensitive, while co-occupancy with CTCF partially protects TBX5-bound TAD boundaries and loop anchors. These results highlight the importance of lineage-restricted TF dosage in cell-type specific 3D chromatin dynamics, suggesting a new mechanism for TF-dependent disease.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. Here, we show that a Tbx5 ⁺ /Mef2cAHF ⁺ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface. Ablation of Tbx5 ⁺ /Mef2cAHF ⁺ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including misexpression of Slit2 and Ntn1 , which encode guidance cues. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify Tbx5 as a candidate selector gene, directing progenitors and regulating essential cues, to pattern a compartment boundary for proper cardiac septation, revealing mechanisms for cardiac birth defects.

While atrial septal defects (ASDs) and atrial fibrillation (AF) present differently, there is evidence that they share some genetic basis. Here, we have used directed differentiation of human induced pluripotent stem cells into atrial or ventricular cardiomyocytes (CMs) to delineate gene regulatory networks (GRNs) that define each identity. We uncovered accessible chromatin regions, transcription factor motifs and key regulatory nodes specific to, or shared by, both CM types, including the transcription factor TBX5, which is linked to genetic susceptibility of ASDs and AF in humans. Complete TBX5 loss resulted in a near absence of atrial CMs, with a concomitant increase in the abundance of other cell types. Reduced dosage of TBX5 in human atrial CMs caused cellular, electrophysiological and molecular phenotypes consistent with features of atrial CM dysfunction. This included dose-dependent aberrant accessibility of many chromatin regions and perturbation of gene regulatory networks of atrial CM identity. These results suggest that, in addition to stemming from ion channel or extracellular matrix dysfunction, atrial diseases such as ASDs or AF may result from disruptions of atrial CM identity.

Dosage-sensitive transcription factors (TFs) underlie altered gene regulation in human developmental disorders, and cell type–specific gene regulation is linked to the reorganization of three-dimensional (3D) chromatin during cellular differentiation. In this work, we show dose-dependent regulation of chromatin organization by the congenital heart disease (CHD)–linked, lineage-restricted TF TBX5 in human cardiomyocyte differentiation. Genome organization, including compartments, topologically associated domains, and chromatin loops, was sensitive to reduced TBX5 dosage in a human model of CHD, with variations in response across individual cells. Cohesin binding was reduced at TBX5-bound enhancer elements in a TBX5 dose-dependent manner, providing a potential mechanism for disrupted loop formation. These results highlight the importance of lineage-restricted TF dosage in cell type–specific 3D chromatin dynamics, suggesting a mechanism for TF-dependent disease.

Long intergenic non-coding RNAs (lincRNAs) have been implicated in aspects of gene regulation, but their requirement for development needs empirical interrogation. To begin to understand the roles lincRNAs might play in heart development, we computationally identified nine murine lincRNAs that have developmentally regulated transcriptional and epigenomic profiles specific to early heart differentiation. Six of the nine lincRNAs had in vivo expression patterns supporting a potential function in heart development, including a transcript downstream of the cardiac transcription factor Hand2 that we named Handlr ( Hand 2 -associated linc R NA), Rubie, and Atcayos . We genetically ablated these six lincRNAs in mouse, which implicated genomic regulatory roles to four of the cohort, However, none of the lincRNA deletions led to severe cardiac phenotypes. Thus, we stressed the hearts of adult Handlr and Atcayos mutant mice by transverse aortic banding and found that absence of these lincRNAs did not affect cardiac hypertrophy or left ventricular function post-stress. Our results support roles for lincRNA transcripts and/or transcription to regulation of topologically associated genes. However, the individual importance of developmentally-specific lincRNAs is yet to be established. Their status as either gene-like entities or epigenetic components of the nucleus should be further considered.