Multi-omics Analysis of Skeletal Muscle Identifies Dysregulation of Hypoxia-Induced Genes in Peripheral Artery Disease [RRBS]

Epigenetic modifications such as DNA methylation play a critical role in hypoxic cell programs. However, no previous studies have investigated the epigenetic regulation of gene expression in peripheral artery disease (PAD), a condition characterized by intermittent ischemia. In this study, we used reduced representation bisulphite sequencing (RRBS) to investigate how PAD affects the DNA methylome in skeletal muscle of PAD patients with intermittent claudication (IC) or critical limb ischemia (CLI) compared to non-PAD controls. We also used small and bulk RNA-sequencing (RNA-seq), which allowed for data integration. This multi-omics approach identified metabolic and hypoxia-related genes in PAD skeletal muscle that are regulated at the level of methylation and by various microRNAs. Specifically, binding and expression target analysis (BETA) revealed that epigenetic modifications to the DNA methylome contribute to modulation of the mRNA expression of family with sequence similarity 20, member C (FAM20C) and endothelial PAS domain-containing protein 1 (EPAS1), also known as hypoxia-inducible factor-2alpha (HIF-2α). Finally, we explored the effect of revascularization on the skeletal muscle DNA methylome and transcriptome, which identified the activator protein-1 (AP-1) early response transcription factor Fos proto-oncogene (FOS) as the primary gene influenced by revascularization. These results identify novel hypoxia-related genes regulated by DNA methylation in PAD skeletal muscle, which may provide potential new therapeutic targets. Gastrocnemius muscle specimens were collected from the anteromedial segment of the gastrocnemius, approximately 10 cm distal to the tibial tuberosity yielding a total sample of ~250 mg, from 10 non-PAD controls (Control), 10 patients with symptomatic intermittent claudication, both at baseline (pre-revascularization, IC) and 6 months after revascularization treatment (IC-post), and 10 patients with critical limb ischemia (CLI). Reduced representation bisulphite sequencing (RRBS) was performed on genomic DNA.