Abstract 197: Emerging Role of Toll-like Receptor 4 and Heat Shock Protein 60&70 in Ischaemia-Induced Skeletal Muscle Damage In Vitro
OBJECTIVES The innate immune response contributes to the skeletal muscle damage in patients with critical limb ischaemia (CLI); however, the detailed signaling mechanisms are not fully understood. We hypothesized 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 HSP60&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 HSP60&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 HSP60&70, IL6, TNF-α and cleaved caspase-3 (P<0.05).
CONCLUSIONS Increased cytokine release, apoptosis and expression of HSP60&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 HSP60&70 expression. This suggests a potential pathway where TLR4 and its endogenous ligands contribute to a positive feedback loop to maintain a proinflammatory environment during ischaemia.
- © 2013 by American Heart Association, Inc.