Abstract 123: Ischemia Mediates Myogenic Progenitor Cell Dysfunction

Abstract

Introduction Treatment options for critical limb ischaemia (CLI) are limited. Recent evidence has suggested that even with successful revascularisation, patients often show little functional improvement. This has been attributed to a musculopathy that occurs in CLI. Myogenic progenitor satellite cells (SCs) provide skeletal muscle with an intrinsic ability to regenerate. It has been shown that there is an increase in SCs in ischaemic muscle, however their function in ischaemia is poorly understood and we hypothesize that ischaemia has a detrimental effect on SC function. Methods Gastrocnemius muscle biopsies were taken from CLI patients and compared with non ischaemic control biopsies. The phenotypical changes and frequency of satellite cells were investigated using PAX 7 immunohistochemistry and western blot. C2C12 myoblasts were used in vitro, to investigate the effect of ischaemia on muscle progenitor cell function. Myoblasts were exposed to simulated ischaemia for 24, 48 and 72hrs. Proliferation rates were assessed using an MTT assay. Differentiation and apoptosis were assessed by MYOD and cleaved caspase 3 western blotting respectively. Results There is an increased expression of PAX 7 in CLI muscle biopsies, shown by both immunostaining and western blot analysis, suggesting an increased number of SCs in ischaemic human skeletal muscle (p<0.05). Myoblasts cultured in ischaemic conditions demonstrated decreased cell proliferation, reduced myogenic differentiation (decreased MYOD expression), and increased apoptosis (increased cleaved caspase 3 expression). Conclusion Despite an upregulation of SCs in ischaemic tissue, their function is suppressed in ischaemic conditions and this may be contributing to the poor functional recovery of patients post revascularisation. Enhancement of muscle regeneration in ischaemia may be a useful therapeutic adjunct in the treatment of CLI.