Integrated Cardiac Methylome Analysis

Integrated analysis of the 450K and EPIC Bead Array data was conducted using Bioconductor package ChAMP in R (60 (link)). The analysis pipeline has been summarized in Supplemental Figure 1. Briefly, raw methylation data were imported into R and normalized using BMIQ. Then, 450K and EPIC data were merged on common probes and corrected for batch effects using combat function. Additional filtering was performed for common SNPs. DMPs were identified using a cutoff of a 10% change in methylation and q value less than 0.05. The first analysis focused on HF etiology and identified DMPs associated with ICM (ICM versus NF) and NICM (NICM versus NF) using data from pre-LVAD tissue obtained from 36 patients and 7 NF controls. The second analysis focused on the impact of mechanical unloading and identified DMPs that are associated with HF (pre-LVAD versus NF) and RR (post-LVAD versus pre-LVAD) in 8 paired cardiac tissue samples. The variance in global DNA methylation between subjects was assessed using PCA plots using the first 3 principal components. Hierarchical clustering of subjects was performed using the complete linkage method. DMPs common to ICM and NICM were screened for reciprocal changes in gene expression. RNA-Seq data from LV samples of 50 patients with HF (13 with ICM and 37 with NICM) and 14 NF controls was obtained from the Gene Expression Omnibus (GEO), accession number 116250, and analyzed using the limma package in R. A total of 36,755 transcripts with RPKM level of 0 in more than 50% of patients were filtered out. Data were log transformed using (RPKM + 1). Differential expression analysis was performed using linear model 1-way ANOVA. Differentially expressed genes (DEGs) were defined as genes with a P value adjusted for Benjamini-Hochberg FDR less than or equal to 0.05 between HF and control samples. Genes with an absolute log₂FC difference greater than 0.25 were also included for DNA methylation versus gene expression correlation discovery analysis. The locations of human cardiac super enhancers were acquired from previous publications (61 (link), 62 (link)). ChIP-Seq data are available through the GEO using the following accession numbers: adult human heart H3K27ac (GSE101345), adult human heart H3K4me1 (GSE101156), adult human heart H3K27me3 (GSE101387), and adult human heart CTCF (GSE 127553). DMPs located within intergenic regions were mapped to lncRNAs using GENCODE annotation database (63 (link)). LINC00881 protein interactions were retrieved from the RNAct database using the catRAPID algorithm (64 (link)).