Abstract 278: Signaling Pathways Involved in the Cytoprotective Effects of Erythropoietin and Erythropoietin Derivatives in Ischemic Human Myotubes
Objectives: Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously shown presence of the EpoR in human skeletal muscle. Here we aim to show the direct interaction of the EpoR with the βcR. Further, we investigate the potential 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: Ethical approval and informed consent was obtained for gastrocnemius biopsies. Immunohistochemistry and western blot analysis were performed to demonstrate expression of EpoR and βcR in human skeletal muscle. Co-immunoprecipitation (Co-IP) was performed to demonstrate heterodimerisation of the EpoR with βcR. Human myoblasts were isolated from muscle biopsies to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitor, wortmannin, was then 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: The presence of both EpoR and βcR in human skeletal muscle has previously been demonstrated. We have now confirmed heterodimerisation of EpoR-βcR complex by Co-IP. Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. 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.
Conclusion: We have demonstrated EpoR-βcR heteroreceptor in human skeletal muscle. The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic 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.
- © 2012 by American Heart Association, Inc.