Congenital heart disease (CHD) is the most common birth defect, affecting 1% of all live births. While ~90% of patients with CHD survive into adulthood, many comorbidities make CHD an increasingly significant public health problem. Although there is strong evidence of a genetic contribution in up to 90% of CHD, much of this contribution, and its clinical impact, remains poorly understood. The PCGC is the human genetics component of the NHLBI Bench-to-Bassinet Program, and aims to discover the complete repertoire of genes responsible for CHD, identify the specific mutations responsible for CHD in large numbers of participants, and develop genotype-phenotype correlation to determine how genetics influence the clinical outcomes of CHD. Since its inception 14 years ago, the five main and three ancillary PCGC sites have recruited the largest cohort of CHD patients worldwide comprising 17,100 probands with the full range of structural CHD along with 19,291 family members. Genomic data includes whole genome data on 3,010 probands, and exome data on 12,201 probands including 5,707 proband-parent trios. PCGC has expanded available clinical data through the electronic medical record, obtaining diagnosis codes, laboratory values and serial echocardiographic data on 8,588 PCGC probands to date, and linking clinical and genomic data in the HeartSmart datahub. PCGC has found a genetic cause in 35% of CHD and identified sixty statistically-supported core CHD genes. We discovered a significant contribution of chromatin modifier and cilia genes to CHD, and demonstrated the extreme genetic heterogeneity of CHD with ~300 genes contributing to CHD by a dominant mechanism alone. Further, links between the genetic cause of CHD and outcomes including neurodevelopmental sequelae, operative complications, cardiac function and cancer are emerging. Going forward, we anticipate developing a “living” CHD database available to the entire CHD community that enrolls patients from a broad base of centers, obtains genomic data, and connects clinical data to the database longitudinally with the goal of establishing precision medicine for CHD.

Heart valve abnormalities, both inherited and acquired, pose significant health risks ‎globally, affecting millions of individuals. Conditions like rheumatic heart disease and ‎calcific aortic stenosis can lead to reduced quality of life, decreased life expectancy, and ‎even sudden death. While valve replacement with allografts and mechanical or ‎bioprosthetic heart valves is the current standard treatment, they come with ‎complications like blood clot formation and limited durability, particularly impacting ‎pediatric patients. Tissue engineering (TE) has emerged as a promising alternative, ‎offering the potential to develop functional heart valves. When combined with TE, 3D ‎printing enables the creation of complex, patient-specific constructs with the potential ‎to evade the problems of current options. In this work, we have developed composite ‎inks using carrageenan (CG), gelatin, and poly(vinyl alcohol) (PVA) and thoroughly ‎investigated their physical, mechanical, rheological, and biological (in vivo and in vitro) ‎characteristics to confirm their application in TE. Moreover, we have successfully 3D ‎printed tri-leaflet heart valves using these inks in the air with high fidelity when ‎compared to the CAD design. More interestingly, the 3D-printed heart valves were ‎tested in physiological conditions ex vivo, using a pulsatile flow system, and we ‎confirmed that they can withstand aortic conditions. Leaflets' opening and closing ‎during cyclic pulsatile flows were observed, and printed valves could function properly ‎with no regurgitation and mild stenosis.‎ Figure 1. Schematic showing the method used to fabricate the heart valves (top left). ‎Mechanical characterization of the scaffolds (bottom left). Bio- and hemo-compatibility ‎of the scaffolds (top middle). 3D printing of tri-leaflet valves (middle bottom). ‎Histological evaluation of the scaffolds after subcutaneous implantation (top right). ‎Results of the characterization of the valves using a pulsatile flow system (bottom ‎right).‎

Background: Cardiomyopathy is a clinically and genetically heterogeneous heart condition that can lead to heart failure and sudden cardiac death in childhood. While it has a strong genetic basis, the genetic etiology for over 50% of cardiomyopathy cases remains unknown. Methods: In this study, we analyze the characteristics of tandem repeats from genome sequence data of unrelated individuals diagnosed with cardiomyopathy from Canada and the United Kingdom (n = 1,216) and compare them to those found in the general population. We perform burden analysis to identify genomic and epigenomic features that are impacted by rare tandem repeat expansions (TREs), and enrichment analysis to identify functional pathways that are involved in the TRE-associated genes in cardiomyopathy. We use Oxford Nanopore targeted long-read sequencing to validate repeat size and methylation status of one of the most recurrent TREs. We also compare the TRE-associated genes to those that are dysregulated in the heart tissues of individuals with cardiomyopathy. Findings: We demonstrate that tandem repeats that are rarely expanded in the general population are predominantly expanded in cardiomyopathy. We find that rare TREs are disproportionately present in constrained genes near transcriptional start sites, have high GC content, and frequently overlap active enhancer H3K27ac marks, where expansion-related DNA methylation may reduce gene expression. We demonstrate the gene silencing effect of expanded CGG tandem repeats in DIP2B through promoter hypermethylation. We show that the enhancer-associated loci are found in genes that are highly expressed in human cardiomyocytes and are differentially expressed in the left ventricle of the heart in individuals with cardiomyopathy. Interpretation: Our findings highlight the underrecognized contribution of rare tandem repeat expansions to the risk of cardiomyopathy and suggest that rare TREs contribute to ~4% of cardiomyopathy risk.

The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.