Matthew Fincher, David Abraham, George Hamilton, Daryll Baker, Janice Tsui
Royal Free Hospital, London, United Kingdom.
OBJECTIVES: Treatment options for critical limb ischemia (CLI) are limited. Recent evidence has suggested that even with successful revascularization, 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 ischemic muscle, however their function in ischemia is poorly understood and we hypothesize that ischemia has a detrimental effect on SC function.
METHODS: Gastrocnemius muscle biopsies were taken from CLI patients and compared with non-ischemic 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 ischemia on muscle progenitor cell function. Myoblasts were exposed to simulated ischemia for 24, 48 and 72 hrs. 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 ischemic human skeletal muscle (p<0.05). Myoblasts cultured in ischemic conditions demonstrated decreased cell proliferation, reduced myogenic differentiation (decreased MYOD expression), and increased apoptosis (increased cleaved caspase 3 expression).
CONCLUSIONS: Despite an upregulation of SCs in ischemic tissue, their function is suppressed in ischemic conditions and this may be contributing to the poor functional recovery of patients post revascularization. Enhancement of muscle regeneration in ischemia may be a useful therapeutic adjunct in the treatment of CLI.
AUTHOR DISCLOSURES: D. Abraham, Nothing to disclose; D. Baker, Nothing to disclose; M. Fincher, Nothing to disclose; G. Hamilton, Nothing to disclose; J. Tsui, Nothing to disclose.
Posted April 2012