Glutathione depletion affects the expression of genes involved in the molecular adaptation of C2C12 myotubes to contraction : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physiology at Massey University, Auckland, New Zealand

Abstract

Background. Impaired muscle metabolic function and atrophy are closely linked to a wide range of conditions including cancer, diabetes, and aging. Exercise is known to improve muscle metabolism and increase muscle mass, thereby having protective and therapeutic effects against such conditions. During exercise the production of reactive oxygen species (ROS) within muscle increases. These highly reactive molecules are typically balanced by antioxidants and act as signalling molecules bringing about changes in gene expression. However, if the balance between production and detoxification is lost, they can cause oxidative damage. Chronic oxidative stress can cause muscle to atrophy and impair energy metabolism, contributing to the complications of the aforementioned conditions. Glutathione (GSH) is a three amino acid peptide and one of the most abundant antioxidant molecules in muscle, protecting against oxidative damage. In addition, when oxidised by ROS, GSH may interact with and change the activity of a range of other enzymes, affecting the activation of several signalling pathways that influence the expression of genes associated with energy metabolism and antioxidant capacity. This suggests that GSH may play a role in the magnitude of muscle adaptations to exercise through redox sensitive pathways. Aim. The primary aim was to evaluate the involvement of endogenous GSH in the molecular adaptation of skeletal muscle to contractions induced by electrical stimulation (ES) in vitro. Methods. C2C12 myoblasts were cultured and differentiated into myotubes. The experimental groups consisted of controls and those depleted of GSH by a 24-hour treatment with 1 mM buthionine sulphoximine (BSO). These were then submitted to an electrical stimulation protocol of a 1 second pulse every other second of 10 V and 50 Hz for 24 hours. Cell survival was tested using exclusion technique and trypan blue dye to determine if ES or treatment with BSO effected cell viability. Hydrogen peroxide release during ES was measured using a fluorescent probe (Amplex UltraRed©) and GSH content in the myotubes was quantified using a commercial kit. RT-PCR was conducted to measure the expression of key genes associated with muscle adaptation and metabolism following contraction. The genes tested were Catalase, CPT-1B, Citrate Synthase, GLUT4, GPx1, HK2, NRF1. NRF2, PFK, PGC-1α, PPARα, PPARγ, SOD1, SOD2, Tfam, and VEGF-A. Baseline expression was compared to expression immediately following ES as well as 1 and 3 hours after stimulation had ended. Results. The ES protocol nor treatment with BSO, alone or combined, caused any significant change in cell viability when compared to controls. Cellular concentrations of total GSH were successfully depleted in C2C12 myotubes treated with BSO. GSH depletion caused an increase in H₂O₂ concentrations of treated cells, both at baseline and during ES, in comparison to control groups. GSH depletion increased the expression of PPARγ across all time points when compared to control cells and tended to increase expression of multiple genes at baseline when compared to control groups (PFK p = 0.1149, PPARα p = 0.0654, and SOD1 p = 0.086; unpaired t-test). Electrical stimulation upregulated the expression of VEGF, GLUT4, and PGC-1α irrespective of GSH treatment, along with a tendency to increase the expression of CPT-1B (p = 0.0580; two-way ANOVA). Catalase mRNA levels increased over time following ES and were further increased in GSH depleted groups. Discussion. GSH (or the absence of) may affect baseline expression of several genes known to be involved in the molecular adaptation of skeletal muscle to exercise but, following ES, GSH depletion only affected catalase expression. The effects of increased cellular H₂O₂ concentrations, both at rest and during stimulation, along with the activation of signalling pathways by contraction (e.g., MAPK, AMPK, CaMK) are likely to play a role in the changes in mRNA expression seen in this study. This may contribute to the upregulation of genes associated with angiogenesis, fatty acid metabolism, antioxidant potential, and glucose uptake. The increased activity of these signalling pathways is not dependent on GSH or are sufficient to override the lack of GSH. Conclusion. GSH depletion did not impair gene expression following ES. Although changes were seen in the absence of electrical stimulation, these may be due to acute redox signalling. Further investigation into the specific mechanisms is required to identify potential contributing factors, such as altered levels of H₂O₂ and MAPK, AMPK and CaMK signals which arise from exercise, known to modulate gene expression.