Circadian Dysfunction in the Skeletal Muscle Impairs Limb Perfusion and Muscle Regeneration in Peripheral Artery Disease

BACKGROUND: Peripheral artery disease (PAD), caused by atherosclerosis, leads to limb ischemia, muscle damage, and impaired mobility in the lower extremities. Recent studies suggest that circadian rhythm disruptions can hinder vascular repair during ischemia, but the specific tissues involved and the impact on muscle health remain unclear. This study investigates the role of the skeletal muscle circadian clock in muscle adaptation to ischemic stress using a surgical mouse model of hindlimb ischemia. METHODS: We performed secondary analysis of publicly available RNA-sequencing data sets derived from patients with PAD to identify the differential expression of circadian-related genes in endothelial cells and ischemic limb skeletal muscles. We used mice with specific genetic loss of the circadian clock activator, BMAL1 (brain and muscle ARNT-like 1), in adult skeletal muscle tissues ( Bmal1 muscle ). Bmal1 muscle mice and controls underwent femoral artery ligation surgery to induce hindlimb ischemia. Laser Doppler imaging was used to assess limb perfusion at various time points after the surgery. Muscle tissues were analyzed with RNA sequencing and histological examination to investigate PAD-related muscle pathologies. Additionally, we studied the role of BMAL1 in muscle fiber adaptation to hypoxia using RNA and assay for transposase-accessible chromatin with sequencing analyses in primary myotube culture model. RESULTS: Disrupted expression of circadian rhythm–related genes was observed in existing RNA-sequencing data sets from endothelial cells and ischemic limb skeletal muscles derived from patients with PAD. Genetic loss of Bmal1 specifically in adult mouse skeletal muscle tissues delayed reperfusion recovery following induction of hindlimb ischemia. Histological examination of muscle tissues showed reduced regenerated myofiber number and a decreased proportion of type IIB fast-twitch myofibers in Bmal1 muscle mouse muscles in the ischemic limbs but not in their contralateral nonischemic limbs. Transcriptomic analysis revealed abrogated metabolic, angiogenic, and myogenic pathways relevant to hypoxia adaptation in Bmal1 muscle mouse muscles. These changes were corroborated in Bmal1 -deficient cultured primary myotubes cultured under hypoxic conditions. CONCLUSIONS: Circadian clock in skeletal muscle is crucial for the muscle’s response to hypoxia during hindlimb ischemia. Targeting the muscle circadian clock may have therapeutic potential for enhancing muscle response to reduced blood flow and promoting recovery in conditions such as PAD.