Rebekah Yu1, Janice Tsui1, George Hamilton1, David Abraham2, Daryll Baker1
1Division of Surgery and Interventional Science, University College London, London, United Kingdom; 2University College London, Centre for Rheumatology and Connective Tissue Disease, London, United Kingdom.
OBJECTIVES: Erythropoietin (epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of epo and an epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilized in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB).
METHODS: Gastrocnemius muscle biopsies were obtained from patients with critical limb ischemia and control samples were obtained from non-ischemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of epo and ARA-290 on myotubes subjected to simulated ischemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstream (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis.
RESULTS: Exogenous administration of epo and ARA-290 were able to ameliorate the ischemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or epo pre-treated cells, abolished the reduction in apoptosis.
CONCLUSIONS: The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.
AUTHOR DISCLOSURES: D. Abraham: Nothing to disclose; D. Baker: Nothing to disclose; G. Hamilton: Nothing to disclose; J. Tsui: Nothing to disclose; R. Yu: Nothing to disclose.
Posted April 2013