Rebekah Yu, George Hamilton, Daryll Baker, Janice Tsui
Department of Surgery, University College London, London, United Kingdom.
OBJECTIVES: Erythropoietin (Epo) has tissue-protective effects following ischemic injury, mediated through the EpoR-βcR heteroreceptor. Having previously shown presence of the EpoR in human skeletal muscle, we now aim to show the direct interaction of the EpoR with the βcR. Further, we wanted to determine the cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish the role of PI3K/Akt pathway in protecting cells from apoptosis.
METHODS: Gastrocnemius muscle biopsies were obtained from patients with critical limb ischemia and control samples were obtained from non-ischemic patients. Co-immunoprecipitation (Co-IP) was performed to demonstrate heterodimerization of EpoR with βcR. Human myoblasts were isolated to determine the cytoprotective effects of Epo and ARA-290 pre-treatment on myotubes subjected to simulated ischemia. Wortmannin (PI3k inhibitor) was used to determine the role of PI3k/Akt pathway in mediating cytoprotection. 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: EpoR and βcR has been demonstrated in human skeletal muscle. Heterodimerization of EpoR-βcR was confirmed by Co-IP. Epo and ARA-290 were able to ameliorate the ischemia-induced apoptosis on isolated human myotubes. Addition of wortmannin, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Further, a reduction in apoptosis was associated with an increase in phosphorylated-Akt on western blot analysis.
CONCLUSIONS: EpoR-βcR heteroreceptor was demonstrated in human skeletal muscle. ARA-290 attenuates apoptosis in ischemic human myotubes suggesting a potential role in tissue protection during skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.
AUTHOR DISCLOSURES: D. Baker, Nothing to disclose; G. Hamilton, Nothing to disclose; J. Tsui, Nothing to disclose; R. Yu, Nothing to disclose.
Posted April 2012