Abstract 499: Identification and Characterization of Lamin-Associated Domains in Cardiac Myocytes Isolated From Human Patients With Dilated Cardiomyopathy Caused by LMNA Pathogenic Variant and Their Effects on Gene Expression and DNA Methylation

Abstract

Rationale: Lamin A/C (LMNA) is a nuclear inner membrane protein that interacts with genome through Lamin-Associated Domains (LADs) and regulates gene expression. Mutations in the LMNA gene causes a diverse array of diseases that are collectively referred to as laminopathies. Dilated cardiomyopathy (DCM) is a major component of laminopathies and the leading cause of premature death. Objective: To identify and characterize LADs in cardiac myocytes, cardiac DNA methylation status, and transcriptomes and integrate the findings to find the pathogenic pathways in DCM Methods: We performed ChIP-Seq, reduced representative bisulfite sequencing (RRBS), and RNA-Seq in 5 control and 5 DCM hearts, the later with defined pathogenic variants. ChIP-Seq was performed using an anti-LMNA antibody on protein extracts from FACS-isolated myocyte nuclei, identified by expression of PCM1. RRBS was performed on whole heart DNA extracts to identify methylation state of ~ 500,000 CpG sites. RNA-Seq was performed on whole heart ribosome-depleted RNA extracts. LADs were identified using Enriched Domain Detector (EDD). Results: We identified 370±50 LADs with an average size of ~ 1Mbp (range: 0.5 to 10 Mb), collectively covering about 12,000 genes and 12% of the genomic regions in the control myocytes. The number and size of the LADs were unchanged in DCM. However, genomic locations of LADs had shifted, leading to loss of 3,302 genes from the LADs. Integration of LADs, CpG methylation, and transcript levels showed CpG hypermethylation and suppressed transcript levels in the LADs both in control and in DCM hearts. However, the effects were differential and LADs were associated with increased hypermethylation and further suppression of gene expression in DCM hearts. Pathway analysis of integrated omic datasets identified TGFβ1 and cell cycle pathways as the most activated and MPAK and glycolytic pathways as the most repressed pathways in DCM hearts. Conclusions: LADs, CpG methylation and transcriptomes are dysregulated in cardiac myocytes in patients with DCM caused by LMNA pathogenic variants. The dysregulated biological pathways provide potential targets for interventions in DCM caused by LMNA mutations.