A Dissertation Submitted to the Department of Physiology, University of the Witwatersrand, Johannesburg for the Degree of Master of Science. / Extensive disruption of muscle fibres has been shown to occur after short term eccentric exercise where high mechanical forces are generated. This study tested whether downhill running acts as a stimulus for inducing eccentric damage, and results in greater muscle damage and deterioration in muscular performance than an equal workload of uphill running. The study aimed at determining whether an adaptation or training eflect takes place such that the muscle is more resistant to the damaging effects of a repeated bout of the same exercise. In addition, the study aimed at determining whether the lower muscle volumes and forces of muscular contractions in females compared to males, makes females less susceptible to the damaging effects of eccentric contraction. / IT2018

The aim of this project was to determine the role of contraction in skeletal muscle development. The role of the initial spontaneous contractions observed in zebrafish embryos from 17 to 24 hours post fertilisation was examined. Genetic and pharmacologic approaches were used to study paralysis-induced disruption of skeletal muscle structure and function and subsequently determine the role of contraction. The structural and functional characteristics of developing skeletal muscles were found to be regulated by a dual mechanism of both movement-dependent and independent processes, in vivo. Novel data demonstrates that contraction controls sarcomere remodelling, namely regulation of actin length, via movement driven localisation of the actin capping protein, Tropmodulin1. Myofibril length was also shown to be linked to the mechanical passive property, stretch, with lengthening leading to an increase of the muscle’s ability to stretch. In addition, myofibril bundling and the myofilament lattice spacing, responsible for active tension generation via cross-bridge formation, were shown to be unaffected by paralysis and thus, movement-independent processes. Furthermore, the mechanism of the contraction-driven myofibril organisation pathway at the focal adhesion complexes (FAC), was shown to be different in zebrafish compared to mammals, with mechanosensing revolving around the Src protein rather than Fak. In summary, the role of contraction was established as a critical driver of myofibril organisation and passive tension in the developing zebrafish skeletal muscle. Passive tension regulates muscle function by determining its operational range ensuring that the needs of locomotion are met. Furthermore, investigation of FAC’s role in the contractiondriven myofibril organisation pathway led to the discovery of a novel function for Src in zebrafish somitogenesis. These two findings (i) that contraction is a driver of myofibril organisation and (ii) that Src is a key protein of the skeletal muscle development provides the potential for new therapeutic approaches in humans.

En 1997, le caractère culard, un phénotype hypermusclé chez le bovin, est attribué à des mutations dans le gène de la myostatine (MSTN). Depuis, il a été confirmé qu’une baisse de l’activité de la MSTN conduisait à une augmentation de la masse musculaire chez d’autres espèces, y compris chez l’Homme. L’identification de ce facteur et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine comme, par exemple, chez des personnes ayant eu une fonte musculaire importante suite à une immobilisation prolongée ou en conséquence du vieillissement ou d’une maladie chronique. L’objectif majeur de ce projet de recherche a consisté à évaluer de nouvelles stratégies permettant d’augmenter la masse musculaire chez la souris. Pour ce faire, nous nous sommes intéressés à une métalloprotéine de la matrice extracellulaire (MEC), la décorine (DCN), dont l’interaction avec la MSTN a été caractérisée comme étant zinc dépendante. Suite à l’injection de ce Small Leucine Rich Proteoglycan (SLRP) chez la souris dystrophique mdx et Gamma-sarcoglycan-/-, nous avons constaté une augmentation de la masse musculaire consécutive à un phénomène d’hypertrophie associé ou non à de l’hyperplasie. Des études de dose/cinétique ont montré que l’effet positif de la décorine sur la croissance musculaire était maximal 21 jours après administration. Nous avons également découvert qu’un fragment peptidique de 41 acides aminés du domaine N-terminal de la protéine DCN murine conservait une activité anti-myostatine et induisait une hypertrophie musculaire chez la souris dystrophique. Ce domaine, site de l’interaction directe entre la MSTN et la DCN, présente un motif CX3CXCX6C, caractéristique des SLRPs de classe I, dont le cluster de cystéines et son interaction avec le zinc ont été décrits comme indispensables à l’activité anti-MSTN de la DCN. Différentes études concernant les mécanismes induits lors de la séquestration de la MSTN par la DCN dans la MEC ont également été conduites afin d’expliquer les phénomènes observés chez la souris. Enfin, nous avons étudié le potentiel de la DCN pour favoriser la greffe de cellules myogéniques et développé différentes approches de thérapie génique. / In 1997, the double-muscling phenotype, a marked hypermuscularity in cattle, was related to mutations in the myostatin (MSTN) gene. Since, it was confirmed that a decrease of the myostatin’s activity drives an increase of the muscular mass in others species, including Human. The identification of this factor and the consequences of its invalidation on the muscular development open many perspectives in human medicine, as, for example, for people whom have an important muscular loss fallow up an extended immobilization or in consequence of old age or a chronic disease. The main purpose of this research project was to evaluate some new strategies permitting the increase of the muscular mass in mice. To achieve that, we investigated in detail the decorin (DCN), a metalloprotein of the extracellular matrix (ECM), interacting with MSTN in a zinc-dependent manner. After intramuscular injection of this Small Leucine Rich Proteoglycan (SLRP) in mdx and Gamma-Sarcoglycan-/- dystrophic mice, we observed a significant increase of the muscle mass conducted by hypertrophy associated or not with hyperplasia. Dose and cinetic studies showed that the positive effect of the decorin on muscular growth was maximal 21 days after administration. Furthermore, we showed that a peptide encompassing the 31-71 sequence retains full myostatin binding capacity and intramuscular injection of this peptide induces muscle hypertrophy in dystrophic mice. This direct interaction site between MSTN and DCN contains a conserved CX3CXCX6C pattern of class I SLRPs, whose cluster of cysteins and its interaction with zinc were shown to be crucial in the anti-MSTN activity of DCN. Various studies of the mechanism resulting of the sequestration of MSTN by DCN in ECM were conducted in order to explain the phenomenom observed in mice. Al last, we have studied the potential of DCN in the cellular transplantation and developped different anti-myostatin strategies of genetic therapy.

The methodologies utilised by pharmaceutical companies for the toxicity screening of developmental drugs are currently based on outdated two-dimensional (2D) plate-based assay systems. Although such methods provide high-throughput analysis, limitations surrounding the biomimicry of the culture environment reduces the accuracy of testing, making the process cost and time inefficient. To significantly enhance the current methods, a screening platform that is both flexible in its design and is amenable toward physiologically representative engineered tissue is required. Incorporating a flow environment within the system elicits a variety of advantages over standard static cultures, pertinently the ability to couple the flow path with automated analytical systems via the use of intuitive software. Musculoskeletal pathological conditions account for £4.76 billion of NHS spending as of 2011 (Department of Health), affecting one in four of the UK adult population. Skeletal muscle, a highly metabolic and regenerative tissue, is involved in a wide variety of functional, genetic, metabolic and degenerative pathological conditions such as muscular dystrophy, diabetes, osteoarthritis, motor neuron disease and pertinently muscular weakness associated with aging populations. Skeletal muscle tissue engineering is centred on the in vitro creation of in vivo-like tissue within laboratory environments and seeks to aid the development of future therapies, by reliably elucidating the molecular mechanisms that regulate such conditions. However, the translation of such models toward systems amenable to pharmaceutical companies has to date been limited. Microphysiological perfusion bioreactors for in vitro cell culture are a rapidly developing research niche, although state of the art systems are currently limited due to the biologically non-representative 2D culture environment, lack of adaptability toward different experimental requirements and confinement to offline analytical methods. Advancements in additive manufacture (AM), commonly known as three-dimensional (3D) printing has provided a method of production that enables researchers to hold complete design freedom and facilitate customisation of required parts. The low cost, rapid prototyping nature of AM further lends itself toward the development of such technology, with design iterations quickly and easily printed, tested and re-designed where appropriate. Issues do however, currently persist regarding the biological compatibility of printed polymers and functional material properties of parts created. As such, this thesis investigated the use of AM as a rapid and functional prototyping technique to design and develop microphysiological perfusion bioreactors. Here, biocompatibility of candidate polymers derived from commercially available 3D printing processes; fused deposition modelling (FDM), stereolithography (SL), selective laser sintering (LS) and PolyJet modelling (PJM) were elucidated. Following the biological evaluation of these polymers, their suitability, and the applicability of each process in function and manufacture of perfusion bioreactors were assessed alongside the research and development process of system designs. Specifically, attention was afforded to the homeostatic environment within bio-perfusion systems. Once finalised, the biological optimisation of designs; biocompatibility and rates of proliferation in response to the perfusion environment, was undertaken. Protocols were then established for the automated perfusion of skeletal muscle cells in both monolayer and tissue engineered 3D hydrogels. This research outlined significant contributions to the scientific literature in 3D printed polymer biocompatibility, in addition to creating bio-perfusion systems that are adaptable, analytical and facilitate the in situ phenotypic development of physiologically representative skeletal muscle tissue. Polymer biocompatibility elucidated in this work will help to facilitate the wide-ranging use of AM in biological settings. However, advancements in the chemical properties of liquid resins for advanced photo-curable processes remain necessitated for AM to be considered as a primary manufacturing technique in the biological sciences. Furthermore, although systems developed in this work have provided a base technology from which to develop and build upon, significant challenges remain in the integration of tissue engineered perfusion devices within pharmaceutical settings. Although it is plausible that the technology created in its current guise would facilitate the automated generation of skeletal muscle tissue, systems require further development to aid their usability and scale. Furthermore, work is also required to optimise the biological environment prior to mass manufacture. As such, to truly influence the pharmaceutical industry, which has invested so heavily in more traditional screening technology, a system that is all-encompassing in biology, technology and automated analytics is required.

Curcumin is a natural compound that improves blood glucose management. While some evidence from isolated mitochondria indicates that curcumin uncouples electron transport from oxidative phosphorylation, the effects of curcumin on mitochondrial respiration and hydrogen peroxide emission in intact skeletal muscle cells are not known. By assessing rates of oxygen consumption, we demonstrated for the first time that curcumin (40 µM) reduced the mitochondrial coupling efficiency (percentage of oxygen consumption that supports ATP synthesis) of intact skeletal muscle cells. A 30-minute incubation with curcumin decreased mitochondrial coupling efficiency by 17.0 ± 0.4% relative to vehicle (p < 0.008). Curcumin also decreased the rate of hydrogen peroxide emission by 43 ± 13% compared to vehicle (p < 0.05). Analysis of cell respiration in the presence of curcumin revealed a 40 ± 4% increase in the rate of oxygen consumption upon curcumin administration (p < 0.05 compared to vehicle). In additional experiments, no difference in mitochondrial coupling efficiency was observed between vehicle- and curcumin-pretreated cells after permeabilization of cell membranes (p > 0.7). The possibility of synergistic effects between curcumin and ursolic acid, another natural compound that improves blood glucose management, was also examined. Interestingly, ursolic acid (0.12 µM) increased mitochondrial coupling efficiency by 4.1 ± 1.1% relative to vehicle (p < 0.008) and attenuated the effect of curcumin when the two compounds were used in combination (decreased mitochondrial coupling efficiency by 8.0 ± 0.9% compared to vehicle, p < 0.008). These results provide evidence for lower mitochondrial coupling efficiency and hydrogen peroxide emission as possible contributors to the increased glucose uptake and insulin sensitivity of subjects after treatment with curcumin but not ursolic acid. Unless cells are assessed in the intact condition, changes to mitochondrial coupling efficiency after curcumin treatment may go unnoticed.

Background: Exercise in pregnancy is associated with optimized fetal growth; however, the implicated mechanisms remain unknown. We hypothesize that exercise-induced myokines may be acting on the placenta to optimize fetal growth across gestation. Methodology: 1) Circulating profiles of 11 myokines were analyzed in 2nd trimester plasma of women characterized as active (N=14) or non-active (N=16) during pregnancy. 2) First trimester human placental explants (N=5) were treated with SPARC in a dose-dependent manner (0-150ng/ml). Metrics of placental health/function, including GLUT-4 expression/regulation, were assessed. Results: 1) Active women demonstrated an elevation in circulating SPARC compared to non-active women (86±19pg/ml vs. 52±18pg/ml, p=0.0001). 2) Explants treated with SPARC at 100ng/ml demonstrated improved invasion, with improved maximum outgrowth distance (N=3; p=0.0219). Conclusion: SPARC is a myokine that is elevated in the circulation of active pregnant women and is associated with improved placental invasion, suggesting a possible role of SPARC in placentation.

Skeletal muscle atrophy is the most common clinical disorder of skeletal muscle and typically occurs as a secondary consequence of fasting, disuse, acute and chronic illness, and aging. It can lead to prolonged recovery and loss of independent living. Of similar clinical significance, one third of Americans are obese and at risk for metabolic syndrome. Interestingly recent studies have demonstrated that both metabolic syndrome and obesity diminish skeletal muscle strength, power, and endurance. However, there are no effective pharmacological treatments for these debilitating effects on skeletal muscle. This is largely due to the fact that the molecular mechanisms underlying its pathogenesis remain uncharacterized. We have recently identified ursolic acid (UA) as a small molecule inhibitor of muscle atrophy. In the absence of atrophy-inducing stress, UA-supplemented chow elicited muscle hypertrophy with little adiposity in mice. To further evaluate these data, mice were subjected to a high fat diet (HFD) with or without UA supplementation, or a standard chow (SC) control. Our data indicates that UA-supplemented HFD mitigates muscle atrophy and adiposity, while HFD significantly reduces muscle mass compared to SC. Furthermore, mice fed a HFD exhibited increased adiposity and reduced muscle mass, strength, and fiber diameter when compared to SC controls. Molecular analysis revealed diminished protein content and increased triglycerides. Gene expression analysis revealed a reduction in Pgc1α, a critical gene that regulates oxidative metabolism and mitochondrial biogenesis. Additionally, we found decreased expression of hormonal receptors AR, involved in signaling of testosterone, and Thrα, involved in signaling of thyroid hormones. Taken together, these data suggest that alterations in gene expression resulting from diet-induced obesity are an atrophy-inducing stress that may function by disrupting metabolic and hormonal signaling.

Satellite cells are a distinct lineage of myogenic precursors that are responsible for the growth of muscle during post-natal life and for its repair after damage. During muscle growth and regeneration satellite cells are activated in response to growth signals from the environment, which induces the expression of one or both of the two MRFs, Myf-5 or MyoD. Activated satellite cells migrate to the site of injury and proliferate before these transcription factors go on to activate transcription of myogenic genes. The myoblasts can then adopt one of two fates. Some myoblasts initiate terminal differentiation and are able to either fuse into existing myofibres to repair them, or fuse with other myoblasts to form new fibres. Other myoblasts do not differentiate but instead return to quiescence and adopt a satellite cell position on repaired or newly formed fibres. Mighty, a downstream target of myostatin that was discovered by the Functional Muscle Genomics Laboratory has recently been shown to induce cell hypertrophy in cell culture through enhanced differentiation and fusion of myoblasts. Myostatin-null mice have hypertrophic muscles and an improved muscle regeneration phenotype. These mice have also been shown to have higher basal levels of Mighty in skeletal muscle than wild-type mice. In this thesis the expression profile of Mighty during skeletal muscle regeneration was characterised in relation to MyoD. During regeneration Mighty gene expression was induced at day five post-injury in both wild-type and myostatin-null mice. In the myostatin-null mice Mighty gene expression remained elevated at day seven post injury in contrast to the levels in the wild-type, which had decreased at this time point. By day-14 and day-28 post-injury Mighty levels were decreased. The up-regulation of Mighty occurs at the time of peak myotube formation in regenerating skeletal muscle, consistent with a role for Mighty in enhancing differentiation and fusion of myoblasts. The extended up-regulation of Mighty in the myostatin-null muscle may be responsible for the enhanced regeneration phenotype of these mice. Analysis of the myotube and reserve cell populations, which are an in vitro model of satellite cells, from both C2C12 cells and Mighty over-expressing clones (Clone 7 and Clone 11) showed that Mighty expression down-regulates two satellite cell markers, CD34 and Sca-1. Both these molecules have been recently shown to be involved in myoblast fusion and reserve cell specification, although their exact role in these processes is not yet known. Expression of Sca-1 is associated with a slowly proliferating non-dividing state while CD34 is associated with the population of reserve cells that do not fuse when notch signalling is inhibited. The results of this thesis indicate that Mighty over-expression may cause the enhanced fusion phenotype by regulating these two molecules. In conclusion the data in this thesis supports a role for Mighty in the myotube formation phase of regeneration and may be able to enhance regeneration by recruiting more myoblasts to terminal differentiation by altering CD34 and Sca-1 expression.

The decline in fertility in dairy cows is of international concern. Since 1950, milk production demands have increased while first service rates of conception have decreased. It is unclear why fertility has decreased, however current dairy management practice requires cows to be kept on a tight yearly calving schedule to ensure maximum milk production over the lifetime of the cow. The current postulate suggests that this regime places a high metabolic burden on the cows, which in turn requires the breakdown of tissues such as fat and muscle to provide substrates to meet the increased energy demands of lactation. Immediately after calving, dairy cows enter a state of negative energy balance (EB), as they cannot consume enough energy to sustain lactation. During this period of negative EB, fat is mobilised in the form of non-esterified fatty acids to help supply the body with the extra energy it needs, but fat mobilisation decreases after four weeks while cows remain in a state of negative EB for several more weeks. It is unclear whether or not muscle breakdown occurs and plays a role in the restoration of EB in lactating cows during peak lactation. I hypothesized that the breakdown of muscle does occur in cows during peak lactation, and that it occurs to a greater extent in cows producing higher amounts of milk. Dairy cows from three strains, NZL, NZH and OSH, representing cows with differing milk production abilities (low, intermediate and high, respectively), were studied for 12 weeks postpartum. Blood was drawn at weekly intervals and muscle biopsies taken at -1, 1, 4, 8, and 12 weeks postpartum. Analysis of plasma revealed an increase in the abundance of troponin I-fs (a marker of muscle breakdown) over the period of study, suggesting that breakdown of skeletal muscle was occurring. Real-time polymerase chain reaction analysis showed that expression of the ubiquitin-proteasome (UbP) ligases atrogin-1 (atro1) and muscle ring finger protein 1 (murf1) increased initially, but returned to normal levels by four weeks postpartum. Concentration of mRNA of the lysosomal proteases, cathepsin B, D, H and L, did not change over the period of study. Therefore, the UbP pathway may contribute to the breakdown of muscle detected by troponin I-fs in plasma. Proteins involved in translation initiation were examined by Western blotting. The ratio of phosphorylated over total eIF2alpha and 4E-BP1 remained unchanged throughout the study, indicating that the breakdown of muscle was not a result of decreased protein synthesis. However, there was a greater ratio of phosphorylated to total eIF2alpha in NZL cows compared with NZH and OSH, suggesting that protein synthesis was less overall in NZL cows than other strains. Measurement of myosin heavy chain composition indicated there was no change in the abundance of type I and type IIx muscle fibres and plasma myostatin levels did not change over the period of study. However, the OSH cows had less myostatin in their plasma than the NZL and NZH cows, suggesting that there may be inhibition of muscle growth occurring in this strain. The results of this study suggest that breakdown of muscle could be important in restoring the EB in high-producing dairy cows during peak lactation. Upregulation of the UbP pathway during the first four weeks of lactation may contribute to this muscle breakdown. However, it remains unclear what processes then continue to regulate breakdown of skeletal muscle to maintain the elevated abundance of troponin I-fs in plasma from four to 12 weeks postpartum in lactating dairy cows.

Myostatin is a negative regulator of muscle growth. When it is not present or non-functional double-muscling occurs, the primary characteristic of this phenotype being an increase in muscle mass. Another characteristic of double-muscling is an increased proportion of type IIB muscle fibres, which rely on glycolysis as their primary energy source, as opposed to type IIA and type I fibres which rely on oxidative phosphorylation. This switch in muscle metabolism directly impacts on the mitochondria, as mitochondria from glycolytic muscle fibres have been shown to have differences in metabolic activity. The increased proportion of glycolytic muscle fibres present in myostatin knockout animals provides a unique model to investigate alterations in muscle fibre type metabolism. The mouse model of myostatin knockout utilised during this study was generated by genetic deletion of exon three of the myostatin gene. Verification of this knockout was attempted by western blot analysis, but only the latency associated protein (LAP) was detected. Interestingly, the LAP was barely detectable in the knockout muscle suggesting deletion of exon three affects binding of anti-myostatin antibodies to the LAP, as that part of the gene is not deleted. A comparison of the basal mitochondrial stress levels was made, also by western blot analysis. The knockout mitochondria showed no change in levels of heat shock protein 60 or superoxide dismutase 2, indicating that they are not being subjected to any increased stress due to the myostatin knockout phenotype. A comparative proteomics approach was used to detect changes in the mitochondrial proteome of myostatin knockout gastrocnemius muscle to gain clues to how mitochondria from glycolytic muscle fibres differ from those present in oxidative fibres. This was undertaken using two-dimensional electrophoresis (2-DE), in-gel tryptic digests and peptide mass fingerprinting by mass spectrometry. A 2-DE gel protein loading of 220 g was shown to give the best protein spot resolution and the most crucial step in the loading process was found to be the laying of the immobilized pH gradient, which had to be performed very carefully to obtain a consistent loading pattern. This study resolved only around 160 protein spots out of the estimated 1,000 to 2,000 proteins present in the mitochondria. Modulation of six proteins was seen at a plt0.1 level, but were unable to be identified using the current methodology. More abundant mitochondrial proteins were able to be identified, but showed no significant modulation. Malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase, which were identified during this study, have been reported to have decreased activity in mitochondria from glycolytic muscle fibres. This study suggests that the change in activity observed by other researchers is due to inhibition of these enzymes in the glycolytic fibres or activation in the oxidative fibres.