Abstract 146: Activation of the Toll-like Receptor (TLR) Adapter Protein MyD88 Is Detrimental to Skeletal Muscle in Critical Limb Ischemia (CLI)
Objective: CLI is a cause of significant morbidity and mortality in the western world and is associated with skeletal muscle damage and tissue loss, however its pathophysiology is poorly understood.TLRs have been implicated in ischaemia-induced tissue damage. TLR 2 and 6 in particular have been implicated in critically ischaemic muscle. Two signalling pathways exist for the signalling transduction of TLRs: the MyD88-dependent and the MyD88-independent pathways. We aim to investigate which TLR signalling pathways are involved in ischaemia-induced muscle damage. We hypothesize that skeletal muscle ischaemia induces TLR 2/6 heterodimerisation and activation of the MyD88 dependent pathway resulting in cytokine-mediated muscle damage.
Methods: TLR 2/6 expression in ischaemic and control human muscle biopsies and in C2C12 myotubes cultured in ischaemic conditions were studied using Western blot and immunohistochemistry. TLR 2/6 heterodimerisation was confirmed by co-immunoprecipitation. Activation of the signalling pathway was assessed using phosphorylated inhibitor of kappa B (IκBα) expression. Functional effects of TLR 2/6 antagonism and MyD88 inhibition on ischaemia-induced IL-6 release and apoptosis were studied in myotubes incubated with neutralizing TLR 2/6 antibodies or MyD88 inhibitor. IL-6 release was assayed by ELISA. Apoptosis was assessed using cleaved caspase-3.
Results: TLR 2/6 protein expression was significantly upregulated in ischaemic human muscle and ischaemic C2C12 myotubes (p<0.05). TLR 2/6 co-localise and heterodimerise under ischaemic conditions with consequent activation of the signalling pathway as shown by upregulation of phosphorylated IκBα. Ischaemia-induced IL-6 production and apoptosis were reduced by TLR 2/6 antagonism and MyD88 inhibition (p<0.05).
Conclusion: Upregulation of TLR 2/6 expression occurs in CLI. Heterodimerisation of TLR 2/6 and the subsequent activation of the signalling pathway results in IL-6 release and apoptosis which contributes to inflammation and muscle damage in ischaemia. Further we have demonstrated that the MyD88 dependent and not the MyD88 independent pathway is important in the TLR mediated muscle damage and thus this pathway may be a potential therapeutic target.
- © 2012 by American Heart Association, Inc.