Engineered heart tissues facilitate functional characterization of noncoding variants implicated in Hypertrophic and Dilated Cardiomyopathy [Bulk RNA-Seq]

Inherited cardiomyopathies frequently arise from rare, highly penetrant coding variants with variable clinical expressivity. Recent biobank-scale genome-wide association studies (GWAS) suggest significant polygenic contributions to cardiovascular diseases, including cardiomyopathy. Most GWAS loci map to noncoding regions, which are poorly conserved across species, requiring a human genome context for experimental validation. We created engineered heart tissues (EHTs) from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and primary cardiac fibroblasts. We assayed single-cell gene expression and chromatin accessibility to generate comprehensive genome-wide regulatory maps. Open chromatin regions (OCRs) were integrated with chromatin contact information and used to functionally fine-map single nucleotide polymorphisms (SNPs) in cardiomyopathy GWAS. SNPs and their associated regulatory regions were assessed using reporter assays, genome editing, and expression profiling. Single-cell RNA-seq of EHTs confirmed populations recapitulating major cell types found in hearts, with advanced cardiomyocyte maturation compared to monolayer hiPSC-cardiomyocytes. More than 400,000 OCRs were resolved to cell type and assayed for canonical transcription factor footprints. Functional fine-mapping of GWAS loci prioritized 5817 variants, and reporter assays on select variants validated allele-specific enhancer activity. We identified a locus harboring significant GWAS signals from both dilated cardiomyopathy and left ventricle ejection fraction in an intergenic region at chr3p25.1. Several of these variants lie in OCRs participating in long range chromatin interactions with SLC6A6 and GRIP2. Haplotype-resolved and synthetic reporter assays confirmed enhancer activity and narrowed candidate SNPs. CRISPR-deletion of this region reduced expression of both SLC6A6 and GRIP2, indicating the enhancer regulates the expression of more than one gene. EHTs with the enhancer deletion displayed markedly reduced contractile function, confirming the necessity of this enhancer region for normal contractility. Taken together, EHTs derived from hiPSCs are an experimentally tractable platform for testing the function of noncoding variants as modifiers of cardiomyopathy. Variants fine-mapped from cardiomyopathies using EHT regulatory maps have functional consequences and provide a set of prioritized sites to advance the study of polygenic heart failure liability.