Investigating the impact of extracellular heat shock proteins on skeletal muscles; a potential role in muscle dysfunction in the critically ill?

Chaudhry, Ali Hasan (2022) Investigating the impact of extracellular heat shock proteins on skeletal muscles; a potential role in muscle dysfunction in the critically ill? Masters by Research thesis (MPhil), Manchester Metropolitan University.

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Abstract

An ongoing battle in long term hospital care is the onset of ICU acquired weakness, several studies have reported the elevated risk of ICUAW in cohesion with pre-existing medical conditions. Skeletal muscle dysfunction is a hallmark of ICUAW, with several studies reporting mitochondria can play a significant role; however, the mechanisms responsible are poorly characterised. Damage associated molecular patterns (DAMPs) are a broad classification of cellular components, which can modulate inflammatory pathways and are highly prevalent in chronic inflammatory conditions (e.g., sepsis) - one example of this is heat shock protein (HSP) 60. HSP60 functions as a molecular chaperone when in the intracellular and a cytokine when in the extracellular environment. We reasoned that eHSP60 may be relevant to muscle dysfunction in sepsis and looked to evaluate this capability in a pre-clinical in vitro model. This study aimed to evaluate the impact of Extracellular Heat Shock Proteins on C2C12 cells, illustrating the potential effect of, eHSP70 and eHSP60 specifically, on the mitochondria and the effectiveness of TAK242 as an inhibitor of TLR4 which enables the production of lipopolysaccharide induced inflammatory mediators. C2C12 cells were treated with increasing concentration of eHSP70 and eHSP60 (5ng/ml, 10ng/ml, 25ng/ml, and 50ng/ml), with or without TAK242, followed by a 24-hour incubation period. U937 conditioned media was acquired in order to mimic an environment similar to that of an ICU patient, this was done by treating U937 cells with 100ng/ml of LPS for 1 hour followed by 1, 2, 4, 24-hour recovery period, the supernatant was collected and used to treat C2C12 cells. Cells were counted using a Haematocytometer and quantified using WST-8 assay, assessing for cell viability. Mitochondrial function was assessed using Seahorse Extracellular Flux Analysis. Cell viability was seen to reduce when treated with increasing doses of eHSP70 and eHSP60, a significant reduction seen when treated with 5ng/ml of eHSP60. Overall parameters of seahorse extracellular flux analysis were seen to increase overall.