Insights into Cardiomyocyte Biology and Gene Regulation through CRISPR-based Assays

Abstract

Heart disease, including heart failure, is the leading cause of death worldwide. A promising therapeutic approach for treating heart failure is restoring the expression of key genes whose dysregulation accelerates heart failure progression. Despite progress in developing innovative heart disease treatments, significant challenges remain. This dissertation addresses two such challenges: (1) characterizing the regulatory network governing a key dysregulated factor linked to heart disease, and (2) understanding how this network responds to perturbation and what the functional consequences of those perturbations are.First, elucidate the regulatory mechanisms of two key cardiac factors, MYH6 and MYH7, in basal and stressed states resembling early stages of heart failure. To identify critical noncoding regulatory elements involved in regulating key cardiomyocyte genes, we performed bulk ATAC-seq and RNA-seq on iPSC-CMs with and without GSK3 inhibition (GSK3i). Significant changes in chromatin accessibility surrounding the MYH6 and MYH7 loci, accompanied by alterations in gene expression, were observed. These genes are essential for cardiac function, with MYH6 silenced during heart failure and its mutations associated with various forms of heart failure. We identified a regulatory region of interest, R3, and profiled its epigenetic landscape in human and mouse samples. This region interacts with the MYH6 transcriptional start site (TSS) and shows activity in both human and mouse cardiac tissues. Second, we demonstrated the utility of combining CRISPR-epigenome editing at noncoding regulatory elements with other functional assays to assess the role of the noncoding regulatory element in myosin regulation and disease progression. We performed epigenome editing of R3 to assess its effects on target gene expression, downstream effects across other cardiac factors, alteration to the cellular response to stress, and its direct role in local chromatin looping. Activation of R3 led to a significant increase in MYH6 and MYH7 expression. Similar effects were observed upon R3 activation in a mouse cardiomyocyte cell line, HL-1. Increased expression of these two myosin heavy chain isoforms altered cellular responses to the hypertrophic stimulus ET-1, resulting in enhanced reactivation of NPPA and NPPB. We observed that the enhancer-activated cells were functionally distinct from their control counterparts based on immunofluorescent imaging and calcium dynamics in basal and stressed states. Finally, we measured the effects of R3 activity on chromatin looping, finding that repressing or activating R3 affected chromatin contacts associated with MYH6. These changes mirrored chromatin interactions observed across chambers of the human heart, confirming its functional activity in humans. When we stressed iPSC-CMs, contacts between the MYH6 TSS and a previously identified regulatory element, C3, were disrupted; however, R3 activation counteracted this loss. In this study, we characterize the regulatory mechanisms governing MYH6 and MYH7 in basal and stressed states, highlighting the complex interplay of regulatory elements in cardiac gene expression. This work identifies endogenous methods of control for MYH6 and MYH7 that hold potential for future therapeutic applications targeting MYH6 reactivation. In summary, this research addresses a critical challenge in developing cardiac therapies and demonstrates the utility of CRISPR-epigenome technologies for investigating cardiac gene regulation.