Methylation changes in dilated cardiomyopathy (DCM) alter cardiac disease gene expression. We investigated DCM mechanisms influenced by CpG methylation using multi-omics and causal analyses in the largest left ventricular tissue cohort. We mapped DNA methylation at ~850,000 CpG sites in failing and non-failing heart tissue across two independent DCM cohorts (discovery n=329, replication n=85), along with array-based genotyping and RNA sequencing. To explore the causal contribution of sentinel CpGs to DCM, we applied Summary data-based Mendelian Randomization (SMR). Sentinel CpGs were fine-mapped using blood samples from a population-based cohort(n=1974). Coordinated changes across multiple CpG sites were examined using weighted gene correlation network analysis (WGCNA). We identified 194 epigenome-wide significant CpGs associated with DCM (discovery P<5.96E-08), enriched in transcriptionally-active chromatin states in heart tissue. Among the 194 sentinel CpGs, 183 significantly influenced the expression of 849 proximal genes (±1Mb). SMR analyses suggested the causal contribution of two sentinel CpGs to DCM and 36 sentinel CpGs to the expression of 43 unique proximal genes (P<0.05). For three sentinel CpGs, colocalization analyses supported a single causal variant underlying the relationship between methylation and target gene expression. Target genes of these CpGs (IER5, ENPTD6, ABHD12, KCNC4) had functions relevant to DCM pathogenesis, warranting future investigation in a cardiac context. Fine-mapping of regions surrounding DCM sentinel CpGs revealed additional signals linked to cardiovascular traits including hsCRP and blood pressure. Co-methylation modules were enriched in genes related to cardiac development, inflammation, extracellular matrix alterations, muscle adaptation, and metabolism, as well as binding sites for the corresponding transcriptional regulators and effectors of these pathways (THRA, PTRF, NSDD2, HOXA9, LZBTB, RXR, YAP (Hippo pathway); p<0.001). Using the largest series of left ventricular tissue to date, this study investigates the causal role of cardiac methylation changes in DCM and suggests targets for experimental studies to probe DCM pathogenesis.

The authors have declared no competing interest.

This research is supported by a Start-Up Grant (awarded to Marie Loh [PI]) from Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The MAGNet repository includes samples from normal donors at the time of cardiac transplantation. The study protocol was approved by the Institutional Review Board at the University of Pennsylvania, and all patients provided written informed consent to participate.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Correspondence and requests for materials should be addressed to Marie Loh (marie_loh@ntu.edu.sg)