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 PS182. Skeletal Muscle Damage in Critical Limb Ischemia (CLI) is Mediated via the MyD88-dependent Signalling Pathway

​Hemanshu Patel1, George Hamilton1, David Abraham1, Daryll Baker1, Sidney Shaw2, Janice Tsui1
1Division of Surgery & Interventional Science, UCL, London, United Kingdom; 2University of Bern, Bern, Switzerland.

OBJECTIVES: Critical limb ischemia is associated with skeletal muscle damage and tissue loss, however its pathophysiology is poorly understood. Toll-like receptors (TLRs) have been implicated in ischemia-induced tissue damage. TLR 2 and 6 in particular have been implicated in critically ischemic muscle. Two separate signalling pathways exist for the signalling transduction of TLRs: the MyD88-dependent pathway and the MyD88-independent pathway. We aim to investigate which TLR signalling pathways are involved in ischemia-induced muscle damage. We hypothesize that skeletal muscle ischemia induces TLR 2 and 6 heterodimerization and activation of the MyD88-dependent pathway resulting in cytokine-mediated muscle damage.

METHODS: TLR 2 and 6 expression in ischemic and control human muscle biopsies and in C2C12 myotubes cultured in ischemic conditions were studied using Western blot and immunohistochemistry. TLR 2 and 6 heterodimerization was confirmed by co-immunoprecipitation. Functional effects of TLR 2/6 and MyD88 antagonism on ischemia-induced IL-6 release and apoptosis were studied with neutralizing TLR 2/6 antibodies and a MyD88 inhibitor. IL-6 release was assayed by ELISA and apoptosis was assessed using cleaved caspase-3.

RESULTS: TLR 2 and 6 protein expression was significantly upregulated in ischemic muscle and ischemic C2C12 myotubes (p<0.05). TLR 2 and 6 heterodimerize under ischemic conditions with consequent activation of the signaling pathway. Ischemia-induced IL-6 production and apoptosis were reduced by TLR 2/6 antagonism and MyD88 inhibition.

CONCLUSIONS: Upregulation of TLR 2 and 6 expression occurs in CLI. Heterodimerization of TLR 2 and TLR 6 and the subsequent activation of the signaling pathway results in IL-6 release and apoptosis which contributes to inflammation and muscle damage in ischemia. Further we have provided novel evidence that the MyD88 dependent pathway is critical in the signalling process and maybe a potential therapeutic target.

AUTHOR DISCLOSURES: D. Abraham, Nothing to disclose; D. Baker, Nothing to disclose; G. Hamilton, Nothing to disclose; H. Patel, Nothing to disclose; S. Shaw, Nothing to disclose; J. Tsui, Nothing to disclose.

Posted April 2012

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