Doctoral Degrees (Physiological Sciences)

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The contribution of inflammatory mediators to delayed secondary muscle damage
(Stellenbosch : Stellenbosch University, 2013-03) Van de Vyver, Mari; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: Understanding the contribution of divergent individual response patterns remains a key objective in identifying mechanisms of inflammation and potential factors limiting the resolution of inflammation. The purpose of this research project was to investigate downstream effects of inflammation following exercise-induced muscle damage in human subjects. Methods: For three different studies, a total of 53 untrained healthy male participants were recruited and divided into a non-exercising control (n=13) and exercise-induced muscle damage groups (n=40). The study design for the three studies was the same (with few exceptions): Downhill running (DHR) (12 x 5min bouts, 10% decline, 15 km.h-1) with blood samples taken pre, post, after 2 and 4 hours post-exercise (2h, 4h) and on days 1, 2, 3, 4 and 7 (d1-d7). Serum was analysed for creatine kinase activity (CK), myoglobin (Mb), cortisol, cytokine (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokine (G-CSF, MIP-1β) and adhesion factor (sICAM-1, sP-selectin) concentrations. Tissue degradation was assessed by serum matrix metalloprotease (MMP-9) and myeloperoxidase (MPO) content. White blood cell differential count was determined and the surface expression of various cluster of differentiation factors (CD11b, CD163, CD68, CD88, CD34) as well as intracellular MPO were assessed in whole bood using flow cytometry. Nuclear localization of the inflammatory mediator NFĸB in isolated perhipheral blood mononuclear cells (PBMCs) was determined using immunofluorescence microscopy. Muscle biopsies (vastus lateralis) taken at baseline, 4h, d1 and d2 were analysed for fibre type, inflammatory and stress-induced pathways (STAT3, IĸBα, p38MAPK), myogenic factors (MyoD, myogenin), neutrophil activity (MPO) and satellite cell number (Pax7). Results: Participants in the DHR group were subdivided into those with a normal recovery (DHR1) and those who developed secondary damage (DHR2). CK peaked on d1 in both subgroups (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) and again on d4 only in the DHR2 group (1110 ± 184 u.L-1). A similar IL-6 and IL-10 response was evident immediately post DHR in all individuals. Additional IL-6 was released in the DHR2 subgroup peaking at 4h (10.3 ± 4.2 pg.mL-1) whereas IL-10 had returned to baseline. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) and CD163+ (3%) monocytes were significantly higher in the DHR2 subgroup. Neutrophil count at 2h (DHR1: 8.6 ± 0.8 x109 cells.L-1, DHR2: 11.4 ± 1.8 x109 cells.L-1) was significantly (p<0.02) correlated to CK activity on d4. PBMC NFĸB p65 nuclear localization was slightly less at 2h in the DHR2 compared to the DHR1 and control groups. Intramuscular STAT3 signalling and MPO were significantly higher in the DHR2 compared to the DHR1 subgroup at 4h and d2 respectively. The progenitor cell response was similar for all DHR individuals with an increase in Pax7+ SC observed at 4h (0.06 ± 0.01 Pax+ SCs/fibre) and d1 (0.07 ± 0.02 Pax+ SCs/fibre). Conclusion: Healthy young men can be divided into those with a adequate and those with a less efficient capacity to control the post damage inflammatory response. The early cytokine response, especially IL-6, seems to be a key role player in the cascade of events leading to late secondary skeletal muscle damage.
The effect of melatonin treatment on doxorubicin-induced skeletal muscle atrophy within a cancer model
(Stellenbosch : Stellenbosch University, 2018-12) Isaacs, Ashwin Wayne; Engelbrecht, Anna-Mart; Loos, Ben; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background and Aim: Skeletal muscle atrophy is a major concern in patients suffering with malignancy. Chemotherapeutic agents, such as doxorubicin (DOX), can further exacerbate this loss of skeletal muscle. Although many cancer patients on chemotherapeutic agents suffer from this condition, there are no therapies routinely used to moderate muscle atrophy. The aim of the study was to investigate whether melatonin (MLT) can attenuate doxorubicin‐induced skeletal muscle and myotube atrophy in an in vivo rodent model of breast cancer as well as in an in vitro model of DOXinduced myotoxicity respectively. The safe and cost‐effective role of melatonin as a possible therapy to limit the burden of doxorubicin‐induced muscle toxicity in cancer patients serves as rationale for the in vivo study and the in vitro study allows for the exploration of more invasive mechanistic aspects using the cell lines, which would not be possible when viewing excised tissue. Methods: Female Sprague‐Dawley rats were inoculated with LA7 cancer cells and were randomly assigned to six groups: Control, Tumour control (TCON), Vehicle control (VEH), MLT, DOX and DOX + MLT (DM). Prophylactic treatment of MLT (6 mg/kg) was administered in drinking water daily and rats received three intraperitoneal injections of DOX (4 mg/kg, 3 times at 3‐day intervals). Following sacrifice blood samples (whole blood counts) and skeletal muscle tissue were collected for histological, immunoblot, antioxidant capacity and immunofluorescence analyses. Furthermore, C2C12 myoblasts grown to confluency and differentiated into myotubes were pretreated with MLT (50 nM) for 48h followed by DOX treatment (0.8 μM) for 24h. The effect of MLT treatment on C2C12 myotube diameter, mitochondrial reactive oxygen species (mtROS) production, sirtuin levels and autophagy activity was then assessed. Results: DOX treatment significantly reduced animal weight (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) compared to DM weight (281.5 ± 7.11 g vs. 284.0 ± 6.53 g) and gastrocnemius muscle weight (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) and cross sectional area (CSA), while increasing markers of muscle degradation compared to MLT treated groups. Serum myoglobin levels were significantly elevated in the DOX group compared to the DM group (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); while, white & red blood cell counts (WBC & RBC) were significantly decreased in the DOX group compared to the MLT treated groups respectively (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L1, p˂0.0001). Furthermore, MLT treatment significantly increased intramuscular antioxidant capacity, mitochondrial biogenesis and satellite cell number. In vitro DOX treatment resulted in increased myotube atrophy, mitochondrial ROS levels and these effects were significantly reduced with MLT pre‐treatment. Discussion: The improvement in animal weight, muscle to body weight ratio, muscle CSA as well as the reduction in myoglobin levels in the treatment groups compared to the DOX group indicate that MLT protects against DOX‐induced atrophy. Moreover, MLT pre‐treatment improved circulating levels of WBC & RBC compared to the DOX only group and attenuated skeletal muscle atrophy by reducing cell apoptosis and increasing satellite cell number suggesting that MLT assists with muscle repair. The in vitro study indicated that DOX‐induced myotube atrophy was preceded by increases in mitochondrial ROS. Conclusion: Results indicate that pre‐treatment with exogenous MLT protects against skeletal muscle wasting induced by DOX in a pre‐cachectic tumour‐bearing rat model.
Exercise, stress and immune system functional responses
(Stellenbosch : University of Stellenbosch, 2004-12) Smith, Carine; Myburgh, Kathryn H.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Stress related to chronic exercise affects both the immune and endocrine systems, but there are still many issues that are poorly understood, particularly effects of stress on the functional capacity of immune cells. This thesis probed some of these issues using physiological models of physical and psychological stress. Both exercise training stress and chronic psychological stress in human subjects were shown to result in an up-regulation of spontaneous reactivity of white blood cells in vitro, using two different assays, namely a) a peripheral blood mononuclear cell (PBMC) culture assay measuring immune cell responsiveness and b) a relatively new flow cytometry technique for assessing activation status of cells by their expression of the surface marker CD69, in a lymphocyte subpopulation-specific manner. An up-regulation of immune cell activation in the absence of an additional stressor was associated with a decreased capacity to mount a response to a subsequent mitogen stimulus in vitro after chronic psychological stress and acute, extreme exercise stress. Another novel finding was that cortisol high-responders to chronic psychological stress exhibited a higher spontaneous reactivity of both CD4+ and CD8+ lymphocytes when compared to cortisol low-responders. This result indicates that chronic exposure to cortisol may decrease its usual inhibitory effect on spontaneous T lymphocyte responsiveness. After optimisation of an animal model of mild, psychological stress, we demonstrated (using an IL-6 antibody) that IL-6 is necessary for a full-blown cortisol response to chronic, intermittent mild stress. Results also suggest that IL-6 plays a role in regulation of its own secretion by PBMCs in response to a stressor, by maintaining the production of IL-1β in the face of stress. Basal serum corticosterone concentration was shown to be the main determinant of the magnitude of mitogen-stimulated PBMC secretion of IL-6 in vitro in the stress-free controls. However, after blocking of IL-6 in vivo, IL-1β was identified as a major regulator of IL-6 secretion by mitogen-stimulated PBMCs in vitro, independently of the presence or absence of stress. The implications of these novel findings are that proinflammatory cytokines are sensitively regulated during mild stress.Mean serum cortisol concentration at rest was not a useful tool to assess chronic exercise stress after training intervention. However, classification of athletes at baseline into two groups according to their resting serum cortisol concentration illustrated two distinct patterns for the responses of both cortisol and the cortisol:testosterone ratio to chronic stress. These studies on the effects of chronic stress on parameters of the endocrine stress-axis and the immune system led to the following main conclusions: a) chronic exposure to cortisol results in a decreased inhibition of spontaneous immune cell activity at rest, b) this increased spontaneous activation of immune cells at rest in the absence of a stressor, is associated with a suppression of immune capacity to respond to a subsequent challenge, c) the latter finding is not evident under stress-free conditions where cortisol promoted immune cell IL-6 secretion, and d) IL- 1β and IL-6 are involved in the regulation of each others’ secretion.
Faktore wat die prestasie en gesondheid van vroue-atlete kan beinvloed
(Stellenbosch : Stellenbosch University, 2003-12) Strauss, Johannes Albertus de Wet; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Although it is common knowledge that regular exercise has many beneficial effects on the human body, it is also true that many highly competitive athletes neglect their health for the sake of performance. With this as a general objective for the study, women athletes of the Matie Athletics Club were recruited as subjects and were monitored and tested for several health-related parameters. Current results indicate that, although the average total cholesterol (TC) concentrations of the group were within normal ranges, quite a number of the sprint and field athletes had TC values regarded as a cardiovascular risk (> 5.2 mmol.l"). Serum testosterone levels of the sprint and field athletes were also higher than those of the distance athletes, but a correlation between TC and testosterone was not established. In general, cholesterol intake of women athletes was within the recommended daily allowance (RDA) prescriptions. The high-density lipoprotein fraction was also within the norm, but a better chemical pathological range had been expected. All haematological parameters were within the normal ranges of distribution, but the red blood cell count, haemoglobin concentration and hematocrit were on average lower than the standard average for females. Athletes, quite often, have higher plasma volumes than average and this can disguise normal haematological values and is described as sport anaemia. The current study has also indicated an iron deficiency (83% RDA) in the diet of female athletes in general. Thus the relatively low observed red blood cell count could not necessarily be attributed to sport anaemia. The energy intake was also poor and did not comply with the energy needs of the athletes. Bone mineral density (BMD) and plasma electrolytes were normal. Distance athletes had a higher BMD of the hip compared to the lumbar spine area. This is probably related to the stress to the hip associated with running. A correlation was observed between TC and BMD of the hip of eumenorrheal and amenorrheal athletes, which had not been observed before. The influence of the phase of the menstrual cycle on the immune system is controversial, and the results of the thesis confirm those of other studies that indicated no influence. In addition, it has been shown that the exogenous ingestion of glutamine, before the onset of exercise, can increase the plasma concentration thereof, and that the formerly observed decline (also seen in the current study) after intense exercise can be totally neutralized. This had not been reported before. The physiological significance of this has not been established, but the assumption is that a continuous adequate supply of glutamine will benefit the immune cells with regard to its reaction to pathogens. As reported by others, it has been shown that the ingestion of 5% glucose during long duration exercise eases the stress on the immune system, as both leucocytes and cortisol levels were attenuated compared to intake of a placebo. A new discovery, however, was that the ad libitum ingestion of glucose was not enough to produce desired significant results. The importance of this finding may have practical implications with regard to desirable amounts of glucose supplementation during races. In conclusion: Female athletes of club performance level are on general in a healthy condition, but are not excluded from the risk with regard to cholesterol. The screening of TC alone is insufficient with regard to competitive athletes, unless the sub-fractions are screened as well during routine medical examinations. Adjustments with regard to the energy and iron content of the diet are suggested. Supplementation of glutamine and glucose before and during exercise could be beneficial to the immune system. More studies with regard to the association of cholesterol with BMD are recommended.
Fibroblast growth factors, A potential game plan for regeneration of skeletal muscle
(Stellenbosch : Stellenbosch University, 2019-12) Gudagudi, Kirankumar; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: Adult mammalian tissue regeneration recruits progenitor stem cells. In skeletal muscle, these are primary satellite cells. Primary satellite cells can be harvested from muscle tissue to investigate or even use as potential therapeutic application. Satellite cells exist in quiescence in the muscle tissue and only become activated following an insult. Most studies investigating satellite cells in vitro use already activated satellite cells, called myoblasts. Fibroblast Growth Factors (FGFs) are fundamental in embryonic development but also in adult skeletal muscle regeneration from injury or pathology. Understanding the role of specific members of this growth factor family could assist in improving the understanding of their influence on the regeneration sequence in skeletal muscle. Methods: Isolated satellite cells from human muscle biopsies were expanded in vitro creating primary human myoblast (PHM) clones. In order to distinguish the rate of proliferation between different PHM clones, a comparative index (CI) was established using the cell cycle and total RNA data of the two PHM clones. Two distinct index calculation models were also presented to determine if these may distinguish between the two clones with greater sensitivity. Secondly, the quiescent state is an integral part of stem cell regulation, therefore choosing the right protocol for inducing quiescence is important. In this study, two developed protocols were assessed, and a new blended protocol addressing the limitations of both protocols was established. This method involved the use of suspension culture (SuCu) with knock out serum replacement (KOSR). Finally, FGF6 and FGF2, both individually and sequentially, were used to treat quiescent myoblasts to determine their involvement in activation and proliferation with the use of cell cycle analysis and mRNA assessment of ki67, p21, myf5, and MyoD. Results and conclusion: The development of the CI was successful in determining the difference in proliferation rate for the different clones. Suspension culture with KOSR, the blended protocol method, resulted in reduced ki67 expression and improved quiescence compared to both the SuCu or KOSR alone. Unlike FGF2, individual treatment with FGF6 was adequate to activate the quiescent PHMs and aid their re-entry into cell cycle with consistency in all three PHM clones by upregulating ki67 expression. However, FGF2 did impede the cell cycle inhibition factor p21, indirectly influencing proliferation. Sequential treatment of FGF6 and FGF2 allowed to determine whether the sequence of treatment would be important. The potential for significantly improving proliferation was found for the sequence: FGF6 followed by FGF2. The inverse sequential treatment order did not demonstrate any significant effect on both activation and proliferation of the quiescent cells. In conclusion, using clones that were distinctly different as assessed by the comparative index, this thesis illuminates that the two FGF family members investigated, act on cell cycle in different ways, thus would influence their utilization in experimental or therapeutic applications.
Immune and satellite cells : important role players in muscle recovery after injury
(Stellenbosch : University of Stellenbosch, 2011-03) Kruger, Maria Jacoba; Smith, Carine; Myburgh, Kathryn H.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Muscle injuries are associated with changes in skeletal muscle as well as the immune system. All studies investigating possible treatment modalities have found both positive and negative effects on muscle recovery. Since no universally accepted treatment modality exists, this thesis aims to determine whether a plant-derived antioxidant, proanthocyanidolic oligomer (PCO), might prove beneficial as treatment for sports injuries in order for athletes to return to the sports field quicker. The difference in recovery of muscle following both chronic (supplementation started 14 days prior to injury and continued thereafter) and acute supplementation (supplementation started two hours after injury) were also investigated. Both chronic and acute PCO supplementation in a rat hindlimb contusion injury model resulted in earlier muscle recovery, verified by an earlier satellite cell response compared to the placebo group. This effect was most prominent already at the four hour time point following injury, compared to day seven and three after chronic and acute placebo treatment respectively. PCO supplementation also resulted in quicker foetal myosin heavy chain (MHCf) expression compared to placebo treatment. Chronic supplementation specifically resulted in a blunted circulatory pro-inflammatory cytokine response, whilst allowing for a significant increase in IL-10, an anti-inflammatory cytokine, on day three (in the PCO group only). At tissue level, the response of the muscle pro-inflammatory cytokines, TNF- and IL- 6, coincided with the satellite cell response. Macrophage infiltration into the injured muscle also followed a similar pattern to that seen for the pro-inflammatory cytokines. Macrophages invaded the injured area quicker when supplemented with PCO chronically, however, macrophage infiltration could not explain the cytokine response seen with acute supplementation. Both chronic and acute supplementation with PCO was responsible for a severely blunted neutrophil response, a novel finding of this particular antioxidant. The main findings of the in vivo rodent study were that PCO was able to blunt the neutrophil response, whilst allowing for earlier macrophage infiltration. To establish possible mechanisms by which PCO might exert these beneficial effects, further analysis included determining macrophage phenotypes and neutrophil numbers in circulation. An in vitro neutrophil migration assay was also employed to further elucidate PCO’s ability to blunt neutrophil infiltration into the injured area. For this study, conditioned plasma were harvested from experimental animals and added together with neutrophils from control rats and granulocyte colony stimulating factor (G-CSF) to the insert of the migration chamber. A chemotactic factor, N-formyl methionine-leucine-phenylalanine (fMLP), was added to the bottom well and neutrophils were allowed to migrate for two hours. Results from this study indicated that neutrophil migration was attenuated in vitro in the presence of conditioned plasma from PCO supplemented rats only. The studies in this thesis on the effect of PCO on parameters of muscle and the immune system led to the following main conclusions: a) PCO supplementation resulted in earlier muscle recovery as a result of earlier satellite cell activation and MHCf synthesis; b) PCO favours an anti-inflammatory cytokine reaction, whilst blunting the pro-inflammatory cytokine response; and c) PCO blunted the neutrophil response whilst facilitating earlier macrophage infiltration into the injured area. The specific mechanism of action of PCO to blunt the neutrophil response specifically, possibly includes the ability to suppress adhesion molecule expression on the neutrophils themselves. However, this warrants further investigation.
Investigating the diversity of extracellular vesicle microRNA in response to exercise-induced muscle micro-damage: comparison of targeted plasma microRNA to targeted and in-depth analysis of extracellular vesicle microRNA
(Stellenbosch : Stellenbosch University, 2020-12) Lovett, Jason Andrew Charles; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: Skeletal muscle (SkM) damage occurs routinely in sport and is present in acquired and inherited myopathies. Damage is confirmed by invasive muscle biopsy. Molecular events that occur during SkM injury and repair, including localized immune cell involvement, may be reflected in circulation. A detailed understanding is essential to the development of diagnosis and intervention strategies. Extracellular vesicles (EVs) are nanoscale (30 to 1000 nm) mediators of intercellular communication. EVs are released in abundance by all cell types, contain cargo including microRNA, and are stable in circulation. Circulating EV profiles adapt to differing physiological and pathological states. EVs may therefore serve as non-invasive biomarkers of SkM injury and regenerative processes. Aims: To determine the biomarker potential of microRNAs in plasma, large EVs and small EVs in regard to skeletal muscle damage induced by unaccustomed eccentrically biased exercise. Methods: Two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by 9 healthy male volunteers. Blood samples were taken at baseline, 2 and 24 hr post-exercise. Serum creatine kinase (CK) and perceived muscle pain (PMP) served as indirect markers of muscle damage. Canonical myomiRs, SkM- important miRs and immune-important miRs were analysed in 3 avenues of biomarker: plasma, large EVs and small EVs. Large EVs were isolated using a 10 000 G centrifugation step, whilst the subsequent supernatant was used for plasma analysis and qEV size exclusion isolation of small EVs. Small and large EV-enriched isolates were visualized using STEM, and size and numbers were quantified using NTA. Based on NTA results the highest particle fractions (7- 10) from qEV columns were pooled for RNA analysis. qPCR analysis of plasma, large EV and small EV miRs was done with normalization to an exogenous control. Small RNA sequencing was done on 3 randomly selected participants’ small EV samples from baseline and 24 hr. Results: PMP and CK increased post-exercise. Small EVs were confirmed using gel electrophoresis, NTA and STEM. Large EVs were confirmed using NTA and STEM. Both small and large EVs were visualised using STEM. No change in small or large EVs size or number was seen post-exercise. Plasma miR-1, 133a and 206 increased in abundance at 2 hr, with miR-1 and 206 returning to BL levels at 24 hr. Conversely, plasma miR-208b & 499 also increased at 2 hr, but showed a trend toward a sustained increase at 24 hr. miR-31, a SkM-important miR, decreased in small EVs at 24 hr when compared to BL and 2 hr. No change in immune-important miRs was found in any biomarker source. Lastly, small RNA sequencing revealed an increase in 18 miRs in small EV at 24 hr post-exercise, with a concomitant decrease in 10 miRs. Conclusion: Both small EV and plasma miRs changed in response to muscle-damaging exercise. Each exhibited distinct miR changes, and timing of changes as shown by qPCR. miR-31, for example, decreased only in small EVs post-exercise, whereas most myomiRs increased only in plasma. RNA sequencing revealed more small EV miR changes. However, these were not corroborated with qPCR.

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