Integration of deep sequencing data reveals global regulation of gene expression in human cardiac hypertrophy and heart failure by differential methylation, hydroxymethylation and microRNA expression

Purpose: Many pathways and genes involved in hypertrophy and heart failurehave been described in animal models, but how the transition from compensatoryhypertrophy to heart failure is initiated in humans remains unclear. This study aimsto identify global mechanisms of gene regulation in the human heart in responseto biomechanical stress (pressure overload) by analyzing the transcriptomes and comparing the contribution of methylation, hydroxymethylation and ncRNAs inaortic stenosis patients with two disease conditions: compensatory hypertrophy(normal ejection fraction) and heart failure (reduced ejection fraction).Methods: DNA and RNA were extracted from snap frozen human myocar-dial biopsy samples and RNA-sequencing (RNA-seq), methylated DNAimmunoprecipitation-sequencing (MeDIP-seq), hydroxymethylated DNAimmunoprecipitation-sequencing (hMeDIP-seq) and smallRNA-sequencing(smallRNA-seq) was carried out using deep HiSeq high throughput sequenc-ing. By comparing the patients’ data to control samples without heart disease usingve replicates for each group and experiment, differentially expressed genes, differ-entially methylated regions, hydroxymethylated regions and differentially expressedmicroRNAs were identied.Results: As expected, the transcriptomes show a high degree of deregulation.Transcriptionfactor (TF) network analysis suggests that approximately 20% ofcondition specic differential expression might be a result of TF regulation. Whilethe methylome is slightly changed in both conditions, the hydroxymethylome ismassively changed in heart failure suggesting a regulatory contribution to thefailing hypertrophic phenotype. Furthermore, differential expression of microRNAsis increased in heart failure and network and target analysis suggests that almost allcandidates are involved in the regulation of differentially expressed genes in both,the compensatory hypertrophy group and heart failure group.Conclusion: The data presented here suggest a regulatory contribution of hydrox-ymethylation and the regulation by microRNAs during the transition of humancompensatory hypertrophy to heart failure.