Investigating the impact of extracellular vesicles on mitochondrial function in human skeletal muscle cells

Lota, Manraj Singh (2023) Investigating the impact of extracellular vesicles on mitochondrial function in human skeletal muscle cells. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Background: Extracellular vesicles are a class of membrane limited vesicles that withhold the ability to firstly transport and secondly liaise with several cells such as proteins and lipids. There are 3 major classes of extracellular vesicles, those being microvesicles, apoptotic bodies and finally the most notable out the three, Exosomes. Surrounding extracellular vesicles, the main function was thought to be merely the removal of unwanted substances. However, due to its ability to cell to cell communicate, it has been hypothesised that Extracellular vesicles have a key role in both physiological and pathological mechanisms that make up bodily functions. Skeletal muscles are a huge endocrine organ, and its ability isn’t primarily related to its mechanisms found for physical movement but include acting like a storage for substrates key in regulating core body temperatures. Extracellular vesicles have additionally been questioned with playing a part in muscle regeneration as well as repair. Aim: It was to determine whether cells stimulated with Tumour necrosis factor – alpha caused an increase synthesis of extracellular vesicles and the effect the membrane limited vesicle had on skeletal muscle mitochondrial function. Method: Human skeletal muscle cells were cultured in varying concentrations of Tumour necrosis factor – alpha for either 24 or 72-hour incubations with two different growth medias. RNA, DNA and protein quantifications were carried out with Nanodrop. Changes in mitochondrial function were analysed with Seahorse XFP stress test to determine the effects extracellular vesicle release had on factors such as, non-mitochondrial respiration, maximal respiration and the key marker for muscle mitochondrial dysfunction, proton leak. Results: It was possible to determine that as the concentration of Tumour necrosis factor – alpha increased treatment on skeletal muscle cells, it did cause an increase synthesis of extracellular vesicles as a result. Through the identification of key hallmark exosomal markers (CD9 and CD81). Furthermore, RNA and DNA concentrations were altered, in some cases positively but also decreases in RNA and DNA were recorded. Regarding mitochondrial function, increases in proton leak and decreases in ATP production were produced indicating that the increase in extracellular vesicle could lead toward muscle dysfunction. However, the stimulation of extracellular vesicles didn’t completely disrupt mitochondrial function, in some cases it was seen the improve. Conclusion: Collectively, the results attained showed that Tumour necrosis factor – alpha treated skeletal muscle cells did cause an increase in synthesis for extracellular vesicle synthesis as well the release augmented mitochondrial function.