Abstract 17363: Loss of Skeletal Muscle Bmal1 Impairs Limb Perfusion and Muscle Regeneration in a Mouse Model of Peripheral Arterial Disease

Abstract

Introduction: Peripheral arterial disease (PAD) is a growing pathologic condition worldwide and leads to severe myopathy. Traditional risk factors for PAD, including diabetes, smoking, and advanced age, are associated with disrupted circadian clock function. However, the contribution of circadian disruption to PAD remains poorly understood. Hypothesis: This study aims to investigate the role of skeletal muscle clock in the severity of PAD. Methods: We performed femoral artery ligation (FAL) surgery in male adult-life inducible skeletal muscle-specific Bmal1 knockout ( Bmal1 musc ) (n = 6) and control mice (n = 5). We measured limb perfusion with laser doppler imaging weekly and assessed post-surgical angiogenesis with CD31/αSMA co-immunostaining and muscle recovery with hematoxylin and eosin (H&E) staining and Laminin immunohistochemistry at 30 days post-surgery. We also performed RNA-sequencing, qPCR, and Western blotting assays to characterize the differentially expressed genes in both ligated and non-ligated hindlimb muscles. Results: Adult-life deletion of skeletal muscle Bmal1 reduced reperfusion recovery, with an average rate of only 50% by 22 days post-surgery in Bmal1 musc mice compared to approximately 80% in control mice. HIF1α target genes including the pro-angiogenic factor Vegfα were significantly downregulated in Bmal1 musc mice. Conversely, genes enriched in the anti-angiogenic TGF-β signaling pathways were up-regulated in Bmal1 musc mice. Myostatin, a myokine that inhibits muscle growth, was downregulated in Bmal1 musc muscles. H&E staining and Laminin immunohistochemistry revealed significantly less but enlarged newly formed myofibers in the tibialis anterior muscle in Bmal1 musc mice. Conclusions: Skeletal muscle BMAL1 plays a critical role in determining the severity of ischemic muscle injury in a mouse model of PAD. Circadian dysfunction impairs hindlimb ischemia reperfusion, decreases the number of newly formed myofibers, and promotes the formation of enlarged regenerating myofibers, which is however previously reported to be associated with compromised muscle force production. Restore muscle circadian function could be a promising strategy to ameliorate myopathic severity in PAD patients.