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Large-scale population studies highlight the relevance of non-coding regions of the human genome in disease susceptibility. This observation suggests that associated non-coding variation may be involved in the alteration of regulatory elements controlling the expression of genes mechanistically implicated in disease. In the cardiovascular specialty, conditions like pathological cardiac hypertrophy or atrial fibrillation (AF) are paradigmatic examples. However, studies deciphering how specific regulatory changes may be linked to disease risk and progression remain largely unexplored. In the case of AF, a significant portion of loci reported in human genome-wide association studies (GWAS) are located in proximity to transcription factor genes; whereas alterations in epigenetic regulation are mechanistically linked to development of cardiac hypertrophy. These observations reinforce a significant role for regulatory mechanisms in both cardiac arrhythmia and hypertrophy.In particular, for this study we aimed to understand the mechanistic role in disease progression for selected non-coding regions in loci associated to AF and left ventricular wall thickness (LVWT).First, for the AF-associated ZFHX3 transcription factor locus, we interrogated a previously unexplored intronic region in high linkage disequilibrium with AF-associated signals and with potential regulatory activity upon ZFHX3. To do this, we employed an in vitro cellular model of adult atrial muscle derived from induced pluripotent stem cells (iPSCs) and applied a CRISPR/Cas9 approach to genetically delete this candidate region. Homozygous knockout showed significantly upregulated levels of ZFHX3 expression, thus assigning a potential repressive role to the interrogated region. We also reported downstream genome-wide transcriptomic changes consistent with those previously reported in murine models of Zfhx3 loss-of-function. Subsequently, in order to determine the epigenetic mechanisms by which ZFHX3 may directly regulate downstream gene expression, we profiled H3K27ac to assess epigenomic changes predictive of enhancer and promoter activity. Interestingly, knockout cells with increased ZFHX3 activity showed significantly stronger H3K27ac enrichments. This result leads us to hypothesise that ZFHX3 largely acts as a repressive transcription factor through direct binding to epigenomic regions with increased H3K27ac levels. Exploratory transcription factor binding site analysis supported our hypothesis in silico, and delineated a set of putative target genes modulated by ZFHX3 repressive activity.Second, we are interrogating non-coding regions across GWAS loci associated to maximal LVWT, a clinical proxy of pathological cardiac hypertrophy. In this context, we have initially targeted the promoter of MYH7, a sarcomere structural gene with known implications in hypertrophy phenotypes, such as those reported in sarcomere-positive hypertrophic cardiomyopathy (HCM) patients. Our results to date indicate significantly reduced MYH7 expression upon homozygous promoter deletion. We are currently expanding our perturbation experiments to examine candidate regulatory regions and their role in hypertrophic phenotypes. In this regard, we have established an in vitro system to assess the impact of deleted genomic elements on the hypertrophic response to endothelin-1 treatment, using a recently developed protocol for left ventricular differentiation. Further molecular and phenotypic characterisation will allow us to assess whether the interrogated regions underlie gene regulatory activities implicated in cardiomyocyte hypertrophy.In conclusion, our application of targeted CRISPR/Cas9 genetic deletions on in vitro models of human cardiomyocytes provides post-GWAS insights into the role of non-coding regions and gene regulation alterations in cardiovascular disease. Our results to date have revealed a novel regulatory element involved in AF progression through repression of ZFHX3 activity. Future efforts will focus on functionally validating predicted ZFHX3 targets in our model of iPSC-derived atrial cardiomyocytes. This cell line will in turn be used as a negative control for hypertrophy experiments in left ventricular cardiomyocytes to investigate novel regulatory regions genetically associated to maximal LVWT and HCM.