Abstract 148: Critical Role of Toll-Like Receptor 4 in Ischemia-Induced Skeletal Muscle Damage

Abstract

Objectives: Inflammation contributes to skeletal muscle damage in patients with critical limb ischaemia (CLI), in whom patient oriented functional outcomes are poor. Previously, we showed that TLR4, a pattern recognition receptor, is upregulated and activated in skeletal muscle of patients with CLI. This study aimed to investigate whether the TLR4 pathway contributes to skeletal muscle damage in CLI, using in vitro and in vivo models. Methods: Human myoblasts, isolated from skeletal muscle biopsies, were cultured to myotubes and exposed to simulated ischaemia, with or without TLR4 inhibitor pre-treatment. Western blot analyses of P-NFkB (signal-related kinase) and cleaved caspase-3 were carried out on cell lysates to assess downstream signalling activity and apoptosis respectively. Further, hind-limb ischaemia by excision of femoral artery was induced in 12-week old TLR4-/- and wild-type (WT) mice with and without administration of the TLR4 antagonist, LPS-RS (n=18 per group). Systemic levels of IL6 & TNF-α, tissue perfusion, inflammatory damage and apoptosis within the ischaemic muscle were assessed at days 3, 7 and 21 by ELISA, laser Doppler, H&E staining and Fluorescent IHC-P, respectively. Results: Simulated ischaemia in cultured human myotubes was associated with upregulation of TLR4, P-NFkB and cleaved caspase-3. Pre-treatment with TLR4 inhibitor reduced P-NFkB and cleaved caspase-3 expression (P<0.05). In in vivo studies, TLR4-/- mice and mice given LPS-RS exhibited reduced systemic IL6 & TNFα levels (P<0.05) following hind-limb ischaemia. This was associated with improved tissue perfusion recovery (P<0.05), reduced inflammatory cell infiltration and diminished apoptosis (P<0.05) in the ischaemic limb as compared to untreated WT mice at days 3, 7 and 21. Conclusion: TLR4 inhibition prior to ischaemia in human myotubes in vitro, was associated with inhibition of the signalling pathway and reduced ischaemia-induced apoptosis. Further, endogenous deletion and exogenous inhibition of TLR4 in vivo were associated with reduced systemic IL6 & TNF-α levels and skeletal muscle damage following hind-limb ischaemia. These findings suggest that TLR4 plays a key role in ischaemia-induced skeletal muscle damage.