Ali Navi1, Rebekah Yu1, Xu Shi-Wen1, Sidney Shaw2, George Hamilton1, Daryll Baker1, Janice Tsui1
1Surgery, University College London, London, United Kingdom; 2University of Bern, Bern, Switzerland.
OBJECTIVES: The innate immune response contributes to the skeletal muscle damage in patients with critical limb ischaemia (CLI); however, the detailed signalling mechanisms are not fully understood. We hypothesised that simulated ischaemia induces inflammatory cytokine release from skeletal myotubes, via a mechanism that involves heat shock protein (HSP) 60 & 70, known endogenous ligands of toll-like receptor 4 (TLR4), in vitro.
METHODS: Human gastrocnemius muscle biopsies were taken from patients with CLI undergoing major lower limb amputation and from patients with no peripheral arterial disease (PAD). Human myoblasts were isolated, cultured to myotubes and then pre-treated with TLR4 neutralizing antibody prior to exposure to simulated ischaemia. Fluorescent immunostaining was carried out to confirm cell differentiation; ELISA analysis were carried out to quantify IL6 and TNF-α release; and Western blot was used to assess expression of HSP 60 & 70, TLR4 and cleaved caspase-3 as a marker of apoptosis.
RESULTS: Myotubes from patients with CLI expressed greater levels of cleaved caspase-3 and TLR4 as compared to those from patients with no PAD. When exposed to ischaemic conditions, increased IL6 and TNF-α release and upregulation of HSP 60 & 70, cleaved caspase-3 and TLR4 were observed in myotubes from both groups of patients compared to culturing in normoxic conditions (p<0.05). Pre-treatment of myotubes from patients with CLI with TLR4 neutralizing antibody prior to simulated ischaemia was associated with reduced expression of HSP 60 & 70, IL6, TNF-α and cleaved caspase-3 (p<0.05).
CONCLUSIONS: Increased cytokine release, apoptosis and expression of HSP 60 & 70 and TLR4 occur in ischaemic skeletal muscle in vitro. TLR4 antagonism was associated with reduced apoptosis and inflammatory cytokine release and down-regulation of HSP 60 & 70 expression. Here we report an intimate link between HSP 60 & 70-induced inflammatory cytokine production and TLR4 inhibition in ischaemic human myotubes.
AUTHOR DISCLOSURES: D. Baker: Nothing to disclose; G. Hamilton: Nothing to disclose; A. Navi: Nothing to disclose; S. Shaw: Nothing to disclose; X. Shi-Wen: Nothing to disclose; J. Tsui: Nothing to disclose; R. Yu: Nothing to disclose
Posted April 2013