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An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology
Thesis (PhD)--Stellenbosch University, 2026.
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Stellenbosch : Stellenbosch University
2026
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| _version_ | 1867613957817106432 |
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| access_status_str | Open Access |
| author | Wentley, Garth Nicholas |
| author2 | Maarman, Gerald |
| author_browse | Maarman, Gerald Wentley, Garth Nicholas |
| author_facet | Maarman, Gerald Wentley, Garth Nicholas |
| author_sort | Wentley, Garth Nicholas |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2026. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/135560 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:44:24.378Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2026 |
| publishDateRange | 2026 |
| publishDateSort | 2026 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/135560 An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology Wentley, Garth Nicholas Maarman, Gerald Hanser, Sidney Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology. Thesis (PhD)--Stellenbosch University, 2026. Wentley, G. N. 2026. An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology. Unpublished doctoral dissertation. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/91020569-252d-4fe5-bd68-56b94989634b Background: Sports injuries are often underpinned by elevated oxidative stress and mitochondrial dysfunction. Owing to Melatonin’s (MEL) ability to localise within the mitochondria and act as a potent antioxidant, it has been proposed as an alternative means of treatment for oxidative stress, mitochondrial dysfunction and injury-related damage to skeletal muscle. However, despite its promising therapeutic potential, much remains unknown with regard to its impact on healthy or pathologically challenged skeletal muscle. Principle Findings for paper 1: MEL at the higher dosage triggers an oxygen-sparing effect on mitochondrial respiration, which is mediated via its antioxidant actions and its ability to enhance pro-survival signalling pathways. For paper 2: MEL improves mitochondrial oxygen consumption, while upregulating ATP levels and altering fission and fusion signalling pathways. For paper 3: MEL preserves and attenuates mitochondrial oxygen consumption and OXPHOS in myotubes by limiting oxygen wastage via an oxygen-sparing action. Moreover, in the pre and curative setting, MEL upregulated mitochondrial fusion. General Methods: For papers 1-3, we have employed an in vitro approach, where differentiated C2C12 myotubes were either unexposed to pathology, or exposed to excess fructose (FRUC) or excess uric acid (UA) and MEL (in different dosages and treatment regimens). For paper 1, Healthy differentiated C2C12 myotubes exposed to MEL. Differentiated C2C12 myotubes were exposed to complete media vehicle control (VC) for 72 hours, 10 nM MEL (72 hours) or 50 nM MEL (72 hours). For paper 2, differentiated C2C12 myotubes were divided into the following experimental groups: Control (CON, receiving only differentiation medium), FRUC (15 mM), and FRUC (15 mM) + MEL (10 nM) for 72 hours. For paper 3, we first followed a pre-treatment regimen: differentiated C2C12 myotubes were either exposed to 10nM MEL or 50nM MEL for 72 hours then exposed to excess UA (750μM for 72 hours), and subsequently, a curative treatment regimen: where differentiated C2C12 myotubes were exposed to excess UA (750μM for 72 hours), briefly washed with PBS and further exposed to either 10nM MEL or 50nM MEL for an additional 72 hours. For co-treatment, differentiated myotubes were divided into control (CON, receiving only differentiation medium), UA (750 μM) and UA + MEL (UA, 750 μM + MEL, 10 nM) groups and were treated for a period of 72 hours. In summary, the following experimental techniques were used in the assessment of our endpoints, including: mitochondrial respiration (Oroboros O2K), ATP (luminescent assay), autophagy and citrate synthase (fluorescent assays), SOD and CAT (spectrophotometric assays) and protein signalling analysis (Western blotting). Results: For paper 1, Here, we exposed healthy, differentiated C2C12 myotubes to two MEL concentrations (10nM or 50nM). The 10 nM concentration did not affect any of the mitochondrial respiration parameters. At a 50 nM concentration, mitochondrial complex II-linked oxidative phosphorylation (OXPHOS), electron transfer system (ETS) capacity, the contribution of complex II to ETS, and residual oxygen consumption (ROX) were reduced. Neither concentration was able to influence the mitochondrial coupling control ratios, nor the coupling control efficiency ratios. Furthermore, both concentrations were unable to influence ATP production but did reduce superoxide dismutase activity. 50 nM increased catalase activity without affecting autophagy or citrate synthase activity. Moreover, 50 nM reduced the expression of activated JAK2 and STAT3 proteins, while 10 nM reduced JAK2 expression without affecting the expression of activated JAK2 and STAT3 proteins. 50 nM increased activated AKT and ERK1/2 expression with no effect on p38 or PGC1-α expression. For paper 2, Here, we exposed differentiated C2C12 myotubes to an excess of FRUC (15 mM) and a regimen co-treatment comprising excess FRUC (15 mM) and MEL (10 nM). MEL co-treatment normalised mitochondrial parameters, including leak respiration, residual oxygen consumption, TMPD-linked respiratory flux, and mitochondrial coupling efficiency. Co-treatment also increased ATP and the activities of CAT and SOD. Co-treatment did not affect caspase-3 protein levels, but it did increase the expression of Drp1 and BAX, potentially priming the tissue for future apoptotic events, while decreasing Opa1 protein expression. For paper 3, Pre-treatment with MEL (after excess UA as a pathological stressor) increased routine respiration (to above normal levels), leak control ratio, complex-1 and 2 linked OXPHOS, ETS capacity, the contribution of complex-2 to ETS, ROX and TMPD flux parameters. Pre-treatment also normalised the OXPHOS control ratio and ETS coupling efficiency. Furthermore, regarding protein signalling, pre-treatment with MEL increased the expression of proteins involved in mitochondrial dynamics, including Drp1, Opa1, MFN1, and MFN2. Lastly, pre-treatment did not affect the activities of ATP, SOD, and CAT. It also did not affect BAX protein expression. MEL curative treatment (after excess UA as a pathological stressor), increased routine respiration and leak, normalised complex-2 linked OXPHOS, ETS capacity, ROX and OXPHOS coupling efficiency. Moreover, regarding protein signalling, MEL curative treatment increased the expression of specific proteins involved in mitochondrial dynamics, including Drp1, MFN1, and MFN2. Furthermore, with the higher concentration of MEL increased BAX protein expression. Moreover, curative treatment decreased Opa1 protein expression, which was further involved in decreasing both SOD and CAT activities in the lower MEL concentration group and only reduced CAT activity in the higher MEL concentration group. Moreover, pre-treatment did not affect ATP. During co-treatment, myotubes were exposed to excess UA (750 μM) and a co-treatment regimen comprising excess UA (750 μM) and MEL (10 nM). Excess UA and MEL co-treatment exposure both drastically decreased ATP levels. Moreover, co-treatment decreased Drp1 expression and normalised Opa 1 expression. Furthermore, excess UA exposure decreased SOD activity and increased Opa1 protein expression. Conclusions: For paper 1, as stated in the results above we conclude that MEL (50 nM) triggers an oxygen-sparing effect on mitochondrial respiration, which is mediated via its antioxidant actions and its ability to enhance pro-survival pathways. MEL intake may have ergogenic effects on healthy muscles in the absence of pathology; for example, consumption before sporting events or physical exercise may help reduce fatigue. For instance, consuming MEL before participating in sports or physical exercise may help reduce the oxidative stress often associated with such activities. For paper 2, we conclude that MEL may help mitigate the toxic effects of excess FRUC on muscle by improving mitochondrial oxygen consumption and alters fission and fusion signalling. Further research is warranted. For paper 3, we conclude that MEL preserves and attenuates mitochondrial oxygen consumption and OXPHOS in myotubes by limiting oxygen wastage via an oxygen-sparing action. Moreover, MEL upregulated mitochondrial fusion in the pre- and curative settings, which has been previously linked to the enhancement of muscle efficiency and performance in situations where excess UA recovery processes occur within skeletal muscle tissue and neuromuscular junctions, possibly improving muscle efficiency and performance in circumstances of excess UA, such as during periods of intensive exercise. Furthermore, by downregulating apoptosis, MEL further protects and maintains mitochondrial homeostasis. Taken together, the data from these individual papers support MEL as a therapeutic agent against muscle injury/damage. Its beneficial effects are mediated by enhanced antioxidant capacity, improved mitochondrial function and protein signalling pathways. Considering that these data were generated in an in vitro setting, future studies could investigate these actions of MEL in a clinical setting. Doctoral 2026-04-01T13:26:38Z 2026-04-01T13:26:38Z 2026-03 Thesis https://scholar.sun.ac.za/handle/10019.1/135560 en Stellenbosch University 148 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Wentley, Garth Nicholas An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology |
| title | An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology |
| title_full | An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology |
| title_fullStr | An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology |
| title_full_unstemmed | An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology |
| title_short | An investigation into the novel effects of melatonin treatment on differentiated C2C12 myotubes in the absence and presence of pathology |
| title_sort | investigation into the novel effects of melatonin treatment on differentiated c2c12 myotubes in the absence and presence of pathology |
| url | https://scholar.sun.ac.za/handle/10019.1/135560 |
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