Nolan, A (2024) Optimisation of an in vitro electrical stimulation model to investigate the role of the vitamin D receptor in skeletal muscle exercise responses. Doctoral thesis, Liverpool John Moores University.

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Abstract

Vitamin D refers to a group of fat-soluble seco-steroids which interact with a variety of tissues within the human body including skeletal muscle. Vitamin D can be converted to 25[OH]D and its biologically active form 1,25[OH]2D3 through the actions of hydroxylases encoded by CYP27A1 and CYP27B1 (Girgis et al. 2014). The vitamin D receptor (VDR) is also present within skeletal muscle and exerts both genomic and non-genomic signaling responses upon exposure to its ligand 1,25[OH]2D3 (Srikuea et al. 2012). Recent evidence suggests that vitamin D is also implicated in mitochondrial regulation, with reductions in respiration and mitophagy observed in both murine cell and rat VDR knock-down (KD) models (Ashcroft et al. 2020; Bass et al. 2021). VDR protein abundance also gradually declines with age, independent of 25[OH]D status and may contribute to the age-related decline in muscle mass and function (Bischoff-Ferrari, Borchers, et al. 2004). Given that exercise is a known therapeutic to improve aspects of mitochondrial biology, it remains to be determined if the VDR is implicated in regulating exercise induced mitochondrial adaptations. Electrical pulse stimulation (EPS) represents a way in which to study both the acute and chronic effects of exercise in vitro (Nikolić et al. 2017). While there has been an increase in the application of EPS within recent years, there does not exist any defining guidelines or consensus on the optimal parameters to elicit specific exercise-like responses. While exercise training in vivo generates divergent post-exercise responses (i.e., improved cardiorespiratory fitness following endurance training) it is not currently known whether this is possible in vitro. Therefore, to appropriately measure the importance of the VDR on the post-exercise responses to EPS, it first must be determined whether EPS is providing the desired exercise-like response. Therefore, this thesis aimed to determine the suitable EPS parameters to elucidate the role of the VDR in regulating endurance-like exercise induced responses in skeletal muscle. There were three overarching objectives:
1. Identify suitable EPS parameters in C2C12 myotubes to induce post-exercise changes in mitochondrial features (gene expression) and activity (complex activity and mitochondrial membrane potential [ΔΨm]).
2. Determine whether stable knock-down of the VDR in C2C12 myotubes influences cell viability, mitochondrial mass, and activity (complex activity and ΔΨm), and whether it augments EPS mediates changes in mitochondrial function including ROS production and lipid peroxidation.
3. Investigate how knock-down of the VDR influences the cellular metabolome in C2C12 myotubes.

The findings in this thesis demonstrate that 3 hr EPS (10 Hz, 10 V, 1 ms) could induce acute changes in genes associated with mitochondrial biogenesis. However, chronic adaptations to exercise were not observed by any EPS protocol and they subsequently failed to induce changes in mitochondrial mass or complex activity. The lack of chronic adaptations is likely due to the longer cell culture time required to observe changes in protein content compared to more rapid changes in gene expression. VDR-KD was associated with reductions in mitochondrial enzyme activity and ΔΨm basally which was independent of changes in mitochondrial content. This aberrant mitochondrial activity was then associated with a greater increase in ROS production and consequent lipid peroxidation following acute EPS treatment. Moreover, VDR-KD resulted in changes in the intracellular metabolome with observed increases in amino acid and purine metabolism. Metabolites associated with oxidative stress such as fumarate and the antioxidant carnosine were also changed in VDR-KD which may help to explain the elevated ROS production present in this phenotype. Taken together, loss of the VDR results in alterations in skeletal muscle mitochondrial complex enzyme activity and ΔΨm. Moreover, VDR-KD is associated with changes in the intracellular metabolome with reductions in antioxidant concentrations. Consequently, this leads to an elevated ROS production following acute EPS. Therefore, VDR signalling is required for the healthy functioning of skeletal muscle mitochondria.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Vitamin D; Muscle; Electrical pulse stimulation; Mitochondria; ROS; Metabolomics
Subjects:	R Medicine > RC Internal medicine > RC1200 Sports Medicine
Divisions:	Sport & Exercise Sciences
SWORD Depositor:	A Symplectic
Date Deposited:	11 Jun 2024 09:16
Last Modified:	11 Jun 2024 09:16
Supervisors:	Owens, D, stuart, C and Close, G
URI:	https://researchonline.ljmu.ac.uk/id/eprint/23390

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