Hypertrophic cardiomyopathy-associated mutations drive stromal activation via EGFR-mediated paracrine signaling.

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7-variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7-variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3-variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.

Engineered human iPS cell models reveal altered podocytogenesis and glomerular capillary wall in CHD-associated SMAD2 mutations.

Early developmental programming involves extensive cell lineage diversification through shared molecular signaling networks. Clinical observations of congenital heart disease (CHD) patients carrying SMAD2 genetic variants revealed correlations with multi-organ impairments at the developmental and functional levels. For example, many CHD patients present with glomerulosclerosis, periglomerular fibrosis, and albuminuria. Still, it remains largely unknown whether SMAD2 variants associated with CHD can directly alter kidney cell fate, tissue patterning, and organ-level function. To address this question, we engineered human iPS cells (iPSCs) and organ-on-a-chip systems to uncover the role of pathogenic SMAD2 variants in kidney podocytogenesis. Our results show that abrogation of SMAD2 causes altered patterning of the mesoderm and intermediate mesoderm (IM) cell lineages, which give rise to nearly all kidney cell types. Upon further differentiation of IM cells, the mutant podocytes failed to develop arborizations and interdigitations. A reconstituted glomerulus-on-a-chip platform exhibited significant proteinuria as clinically observed in glomerulopathies. This study implicates CHD-associated SMAD2 mutations in kidney tissue malformation and provides opportunities for therapeutic discovery in the future.

Predictive modeling of endocardial fibroelastosis recurrence in patients with congenital heart disease.

BACKGROUND: Endocardial fibroelastosis (EFE) is a major effector in the maldevelopment of the heart in patients with congenital heart disease. Despite successful surgical removal, EFE can redevelop, but the underlying cause of EFE recurrence remains unknown. This study aimed to identify hemodynamic predictors and genetic links to epithelial/endothelial-to-mesenchymal transition (EMT/EndMT) alterations for preoperative risk assessment. METHODS: We assessed the impact of preoperative hemodynamic parameters on EFE recurrence in a cohort of 92 patients with congenital heart disease who underwent left ventricular (LV) EFE resection between January 2010 and March 2021. Additionally, whole-exome sequencing in 18 patients was used to identify rare variants (minor allele frequency <10-5) in high-expression heart (HHE) genes related to cardiac EMT/EndMT and congenital heart disease. RESULTS: EFE recurred in 55.4% of patients, within a median of 2.2 years postsurgery. Multivariable analysis revealed specific hemodynamic parameters (mitral valve inflow and area, LV filling pressure, and aortic valve gradient and diameter) as predictors, forming a predictive model with an area under the receiver operating characteristic curve of 0.782. Furthermore, 89% of the patients exhibited damaging variants in HHE genes, with 38% linked to cardiac EMT/EndMT Gene Ontology processes and 22% associated with known congenital heart disease genes. Notably, HHE genes associated with cardiac EMT/EndMT were significantly associated with faster EFE recurrence in a multivariate analysis (hazard ratio, 3.56; 95% confidence interval, 1.24-10.17; P = .018). CONCLUSIONS: These findings established a predictive scoring system using preoperative hemodynamic parameters for EFE recurrence risk assessment. Alterations in HHE genes, particularly those linked to cardiac EMT/EndMT, exacerbate the risk of recurrence.

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury.

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms of myocardial injury in SLE remain poorly understood. In this study, we engineered human cardiac tissues and cultured them with IgG from patients with SLE, with and without myocardial involvement. IgG from patients with elevated myocardial inflammation exhibited increased binding to apoptotic cells within cardiac tissues subjected to stress, whereas IgG from patients with systolic dysfunction exhibited enhanced binding to the surface of live cardiomyocytes. Functional assays and RNA sequencing revealed that, in the absence of immune cells, IgG from patients with systolic dysfunction altered cellular composition, respiration and calcium handling. Phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. Coupling IgG profiling with cell surfaceome analysis identified four potential pathogenic autoantibodies that may directly affect the myocardium. Overall, these insights may improve patient risk stratification and inform the development of new therapeutic strategies.

Endocardial primary cilia and blood flow are required for regulation of EndoMT during endocardial cushion development.

Blood flow is critical for heart valve formation, and cellular mechanosensors are essential to translate flow into transcriptional regulation of development. Here, we identify a role for primary cilia in vivo in the spatial regulation of cushion formation, the first stage of valve development, by regionally controlling endothelial to mesenchymal transition (EndoMT) via modulation of Kruppel-like Factor 4 (Klf4) . We find that high shear stress intracardiac regions decrease endocardial ciliation over cushion development, correlating with KLF4 downregulation and EndoMT progression. Mouse embryos constitutively lacking cilia exhibit a blood-flow dependent accumulation of KLF4 in these regions, independent of upstream left-right abnormalities, resulting in impaired cushion cellularization. snRNA-seq revealed that cilia KO endocardium fails to progress to late-EndoMT, retains endothelial markers and has reduced EndoMT/mesenchymal genes that KLF4 antagonizes. Together, these data identify a mechanosensory role for endocardial primary cilia in cushion development through regional regulation of KLF4.

Genome Sequencing is Critical for Forecasting Outcomes following Congenital Cardiac Surgery.

While genome sequencing has transformed medicine by elucidating the genetic underpinnings of both rare and common complex disorders, its utility to predict clinical outcomes remains understudied. Here, we used artificial intelligence (AI) technologies to explore the predictive value of genome sequencing in forecasting clinical outcomes following surgery for congenital heart defects (CHD). We report results for a cohort of 2,253 CHD patients from the Pediatric Cardiac Genomics Consortium with a broad range of complex heart defects, pre- and post-operative clinical variables and exome sequencing. Damaging genotypes in chromatin-modifying and cilia-related genes were associated with an elevated risk of adverse post-operative outcomes, including mortality, cardiac arrest and prolonged mechanical ventilation. The impact of damaging genotypes was further amplified in the context of specific CHD phenotypes, surgical complexity and extra-cardiac anomalies. The absence of a damaging genotype in chromatin-modifying and cilia-related genes was also informative, reducing the risk for adverse postoperative outcomes. Thus, genome sequencing enriches the ability to forecast outcomes following congenital cardiac surgery.

CHD4 and SMYD1 repress common transcriptional programs in the developing heart.

Regulation of chromatin states is essential for proper temporal and spatial gene expression. Chromatin states are modulated by remodeling complexes composed of components that have enzymatic activities. CHD4 is the catalytic core of the nucleosome remodeling and deacetylase (NuRD) complex, which represses gene transcription. However, it remains to be determined how CHD4, a ubiquitous enzyme that remodels chromatin structure, functions in cardiomyocytes to maintain heart development. In particular, whether other proteins besides the NuRD components interact with CHD4 in the heart is controversial. Using quantitative proteomics, we identified that CHD4 interacts with SMYD1, a striated muscle-restricted histone methyltransferase that is essential for cardiomyocyte differentiation and cardiac morphogenesis. Comprehensive transcriptomic and chromatin accessibility studies of Smyd1 and Chd4 null embryonic mouse hearts revealed that SMYD1 and CHD4 repress a group of common genes and pathways involved in glycolysis, response to hypoxia, and angiogenesis. Our study reveals a mechanism by which CHD4 functions during heart development, and a previously uncharacterized mechanism regarding how SMYD1 represses cardiac transcription in the developing heart.

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury.

Systemic lupus erythematosus (SLE) is a highly heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms by which myocardial injury develops in SLE, however, remain poorly understood. Here we engineered human cardiac tissues and cultured them with IgG fractions containing autoantibodies from SLE patients with and without myocardial involvement. We observed unique binding patterns of IgG from two patient subgroups: (i) patients with severe myocardial inflammation exhibited enhanced binding to apoptotic cells within cardiac tissues subjected to stress, and (ii) patients with systolic dysfunction exhibited enhanced binding to the surfaces of viable cardiomyocytes. Functional assays and RNA sequencing (RNA-seq) revealed that IgGs from patients with systolic dysfunction exerted direct effects on engineered tissues in the absence of immune cells, altering tissue cellular composition, respiration and calcium handling. Autoantibody target characterization by phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. By coupling IgG profiling with cell surface protein analyses, we identified four pathogenic autoantibody candidates that may directly alter the function of cells within the myocardium. Taken together, these observations provide insights into the cellular processes of myocardial injury in SLE that have the potential to improve patient risk stratification and inform the development of novel therapeutic strategies.

The BMAL1/HIF2A heterodimer modulates circadian variations of myocardial injury.

Acute myocardial infarction stands as a prominent cause of morbidity and mortality worldwide1-6. Clinical studies have demonstrated that the severity of cardiac injury following myocardial infarction exhibits a circadian pattern, with larger infarct sizes and poorer outcomes in patients experiencing morning onset myocardial infarctions7-14. However, the molecular mechanisms that govern circadian variations of myocardial injury remain unclear. Here, we show that BMAL114-20, a core circadian transcription factor, orchestrates diurnal variability in myocardial injury. Unexpectedly, BMAL1 modulates circadian-dependent cardiac injury by forming a transcriptionally active heterodimer with a non-canonical partner, hypoxia-inducible factor 2 alpha (HIF2A)6,21-23, in a diurnal manner. Substantiating this finding, we determined the cryo-EM structure of the BMAL1/HIF2A/DNA complex, revealing a previously unknown capacity for structural rearrangement within BMAL1, which enables the crosstalk between circadian rhythms and hypoxia signaling. Furthermore, we identified amphiregulin (AREG) as a rhythmic transcriptional target of the BMAL1/HIF2A heterodimer, critical for regulating circadian variations of myocardial injury. Finally, pharmacologically targeting the BMAL1/HIF2A-AREG pathway provides effective cardioprotection, with maximum efficacy when aligned with the pathway's circadian trough. Our findings not only uncover a novel mechanism governing the circadian variations of myocardial injury but also pave the way for innovative circadian-based treatment strategies, potentially shifting current treatment paradigms for myocardial infarction.

Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease.

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

BACKGROUND: Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. METHODS: We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). RESULTS: From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. CONCLUSIONS: Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

The H2Bub1-deposition complex is required for human and mouse cardiogenesis.

De novo variants affecting monoubiquitylation of histone H2B (H2Bub1) are enriched in human congenital heart disease. H2Bub1 is required in stem cell differentiation, cilia function, post-natal cardiomyocyte maturation and transcriptional elongation. However, how H2Bub1 affects cardiogenesis is unknown. We show that the H2Bub1-deposition complex (RNF20-RNF40-UBE2B) is required for mouse cardiogenesis and for differentiation of human iPSCs into cardiomyocytes. Mice with cardiac-specific Rnf20 deletion are embryonic lethal and have abnormal myocardium. We then analyzed H2Bub1 marks during differentiation of human iPSCs into cardiomyocytes. H2Bub1 is erased from most genes at the transition from cardiac mesoderm to cardiac progenitor cells but is preserved on a subset of long cardiac-specific genes. When H2Bub1 is reduced in iPSC-derived cardiomyocytes, long cardiac-specific genes have fewer full-length transcripts. This correlates with H2Bub1 accumulation near the center of these genes. H2Bub1 accumulation near the center of tissue-specific genes was also observed in embryonic fibroblasts and fetal osteoblasts. In summary, we show that normal H2Bub1 distribution is required for cardiogenesis and cardiomyocyte differentiation, and suggest that H2Bub1 regulates tissue-specific gene expression by increasing the amount of full-length transcripts.

Short-term Pre-operative Methionine Restriction Induces Browning of Perivascular Adipose Tissue and Improves Vein Graft Remodeling in Mice.

Short-term preoperative methionine restriction (MetR) shows promise as a translatable strategy to modulate the body's response to surgical injury. Its application, however, to improve post-interventional vascular remodeling remains underexplored. Here, we find that MetR protects from arterial intimal hyperplasia in a focal stenosis model and adverse vascular remodeling after vein graft surgery. RNA sequencing reveals that MetR enhances the brown adipose tissue phenotype in arterial perivascular adipose tissue (PVAT) and induces it in venous PVAT. Specifically, PPAR-α was highly upregulated in PVAT-adipocytes. Furthermore, MetR dampens the post-operative pro-inflammatory response to surgery in PVAT-macrophages in vivo and in vitro . This study shows for the first time that the detrimental effects of dysfunctional PVAT on vascular remodeling can be reversed by MetR, and identifies pathways involved in browning of PVAT. Furthermore, we demonstrate the potential of short-term pre-operative MetR as a simple intervention to ameliorate vascular remodeling after vascular surgery.

DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

BACKGROUND: Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. METHODS: Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. RESULTS: Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. CONCLUSIONS: Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

Genetic Contribution to End-Stage Cardiomyopathy Requiring Heart Transplantation.

BACKGROUND: Many cardiovascular disorders propel the development of advanced heart failure that necessitates cardiac transplantation. When treatable causes are excluded, studies to define causes are often abandoned, resulting in a diagnosis of end-stage idiopathic cardiomyopathy. We studied whether DNA sequence analyses could identify unrecognized causes of end-stage nonischemic cardiomyopathy requiring heart transplantation and whether the prevalence of genetic causes differed from ambulatory cardiomyopathy cases. METHODS: We performed whole exome and genome sequencing of 122 explanted hearts from 101 adult and 21 pediatric patients with idiopathic cardiomyopathy from a single center. Data were analyzed for pathogenic/likely pathogenic variants in nuclear and mitochondrial genomes and assessed for nonhuman microbial sequences. The frequency of damaging genetic variants was compared among cardiomyopathy cohorts with different clinical severity. RESULTS: Fifty-four samples (44.3%) had pathogenic/likely pathogenic cardiomyopathy gene variants. The frequency of pathogenic variants was similar in pediatric (42.9%) and adult (43.6%) samples, but the distribution of mutated genes differed (P=8.30×10-4). The prevalence of causal genetic variants was significantly higher in end-stage than in previously reported ambulatory adult dilated cardiomyopathy cases (P<0.001). Among remaining samples with unexplained causes, no damaging mitochondrial variants were identified, but 28 samples contained parvovirus genome sequences, including 2 samples with 6- to 9-fold higher levels than the overall mean levels in other samples. CONCLUSIONS: Pathogenic variants and viral myocarditis were identified in 45.9% of patients with unexplained end-stage cardiomyopathy. Damaging gene variants are significantly more frequent among transplant compared with patients with ambulatory cardiomyopathy. Genetic analyses can help define cause of end-stage cardiomyopathy to guide management and risk stratification of patients and family members.

Tbx5 maintains atrial identity by regulating an atrial enhancer network.

Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa , and conversely, increased the expression of ventricular identity genes including Myl2 . Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.

Contribution of Previously Unrecognized RNA Splice-Altering Variants to Congenital Heart Disease.

BACKGROUND: Known genetic causes of congenital heart disease (CHD) explain <40% of CHD cases, and interpreting the clinical significance of variants with uncertain functional impact remains challenging. We aim to improve diagnostic classification of variants in patients with CHD by assessing the impact of noncanonical splice region variants on RNA splicing. METHODS: We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, <2×10-6) variants from 4472 CHD probands in the Pediatric Cardiac Genetics Consortium through a combined computational and in vitro approach. RESULTS: We identified 53 de novo and 74 rare variants in CHD cases that alter splicing and thus are loss of function. Of these, 77 variants are in known dominant, recessive, and candidate CHD genes, including KMT2D and RBFOX2. In 1 case, we confirmed the variant's predicted impact on RNA splicing in RNA transcripts from the proband's cardiac tissue. Two probands were found to have 2 loss-of-function variants for recessive CHD genes HECTD1 and DYNC2H1. In addition, SpliceAI-a predictive algorithm for altered RNA splicing-has a positive predictive value of ≈93% in our cohort. CONCLUSIONS: Through assessment of RNA splicing, we identified a new loss-of-function variant within a CHD gene in 78 probands, of whom 69 (1.5%; n=4472) did not have a previously established genetic explanation for CHD. Identification of splice-altering variants improves diagnostic classification and genetic diagnoses for CHD. REGISTRATION: URL: https://clinicaltrials.gov; Unique identifier: NCT01196182.

Missense Mutation in Human CHD4 Causes Ventricular Noncompaction by Repressing ADAMTS1.

BACKGROUND: Left ventricular noncompaction (LVNC) is a prevalent cardiomyopathy associated with excessive trabeculation and thin compact myocardium. Patients with LVNC are vulnerable to cardiac dysfunction and at high risk of sudden death. Although sporadic and inherited mutations in cardiac genes are implicated in LVNC, understanding of the mechanisms responsible for human LVNC is limited. METHODS: We screened the complete exome sequence database of the Pediatrics Cardiac Genomics Consortium and identified a cohort with a de novo CHD4 (chromodomain helicase DNA-binding protein 4) proband, CHD4M202I, with congenital heart defects. We engineered a humanized mouse model of CHD4M202I (mouse CHD4M195I). Histological analysis, immunohistochemistry, flow cytometry, transmission electron microscopy, and echocardiography were used to analyze cardiac anatomy and function. Ex vivo culture, immunopurification coupled with mass spectrometry, transcriptional profiling, and chromatin immunoprecipitation were performed to deduce the mechanism of CHD4M195I-mediated ventricular wall defects. RESULTS: CHD4M195I/M195I mice developed biventricular hypertrabeculation and noncompaction and died at birth. Proliferation of cardiomyocytes was significantly increased in CHD4M195I hearts, and the excessive trabeculation was associated with accumulation of ECM (extracellular matrix) proteins and a reduction of ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif 1), an ECM protease. We rescued the hyperproliferation and hypertrabeculation defects in CHD4M195I hearts by administration of ADAMTS1. Mechanistically, the CHD4M195I protein showed augmented affinity to endocardial BRG1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4). This enhanced affinity resulted in the failure of derepression of Adamts1 transcription such that ADAMTS1-mediated trabeculation termination was impaired. CONCLUSIONS: Our study reveals how a single mutation in the chromatin remodeler CHD4, in mice or humans, modulates ventricular chamber maturation and that cardiac defects associated with the missense mutation CHD4M195I can be attenuated by the administration of ADAMTS1.