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Cancer cachexia is an unmet clinical need that affects more than half of patients with cancer. Pro-inflammatory cytokines and adipokines could be involved in the pathogenesis and development of cachexia, but this relationship is not completely understood. This thesis aimed to examine and characterise the role of (adipo)cytokines in cancer cachexia. A systematic review was conducted to evaluate the relationship between cytokines and cachexia in people with incurable cancer (Chapter 2). Interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were considerably elevated in cachectic individuals, and a high degree of methodological heterogeneity was observed. Furthermore, data from the REVOLUTION trial were analysed to determine if adiponectin, leptin, intelectin-1 and resistin can predict the modified Glasgow Prognostic Score (mGPS) and cachexia status (Chapter 3). Although adipokines could not predict any of the outcome variables in this study, leptin was negatively associated with mGPS and cachexia, while a positive relationship was identified between resistin and mGPS. Previous research suggested that intelectin-1 might be involved in cancer cachexia. Given the limited availability of literature, a Bayesian meta-analysis was conducted to evaluate the role of intelectin-1 in cancer and its physiological concentration (Chapter 4). Intelectin-1 levels were substantially higher in patients with gastrointestinal cancer compared to controls. The meta-analysis also estimated that the physiological concentration of intelectin-1 ranges from 193ng/ml to 275ng/ml. Lastly, a cell culture model was designed to evaluate the effect of intelectin-1 on human myotubes (Chapter 5). Increased levels of intelectin-1 diminish insulin-mediated glucose uptake and downregulate the expression of genes involved in myogenesis. To conclude, IL-6 and TNF-α were highly expressed in cancer cachexia. Leptin and resistin could also contribute to the development of this wasting syndrome, but to a lesser extent. Besides these (adipo)cytokines, intelectin-1 is another potential biomarker of cachexia and future research should focus on elucidating its mechanisms of action.

Biological rhythms in physiological and behavioural processes anticipate regular environmental changes and therefore adjust physiology and behaviour accordingly. These biological rhythms appear to facilitate the response of skeletal muscle to variable nutrient supply. The timing and/or distribution of nutrients across the day could conceivably alter biological rhythms in this tissue, which could be associated with metabolic health outcomes. However no human research to date has characterised temporal rhythms in in vivo human muscle alongside systemic markers of metabolism. Furthermore, recent studies have revealed links between the first meal of the day and human health, so it is now important to examine the potential underlying mechanisms of these relationships. The aim of this thesis was to take a multi-faceted approach to studying the metabolic and behavioural effects of meal timing, with focus on both absolute and relative timing. Initially, Chapter 4 provided 24-h characterisation of diurnal rhythms in human skeletal muscle gene expression alongside circulating metabolic and endocrine markers. Chapter 5 then built on this by demonstrating the ability of enteral feeding pattern (i.e., continuous versus bolus nutrient delivery) to affect rhythms in skeletal muscle and circulating metabolites - for example, revealing that systemic glucose and NEFA are primarily driven by 24 h profiles of insulin. Chapter 6 confirmed that next morning metabolic control is not affected by hourly sleep fragmentation but is affected when coffee is consumed to remedy a poor night of sleep. The acute experiment described in Chapter 7 demonstrated that enrichment of a typical carbohydrate-rich breakfast with whey protein effectively elicited the second-meal effect but did not differentially alter diet-induced thermogenesis or appetite above that of a carbohydrate-rich breakfast. Finally, Chapter 8 showed that regular daily consumption of that protein-enriched breakfast did not meaningfully alter the major components of energy balance, nor did it change postprandial metabolism or appetite relative either to regular daily consumption of a typical carbohydrate-rich breakfast or daily extended morning fasting. Collectively the research in this thesis highlights the effects of nutrient timing in both absolute terms (i.e., in relation to clock time) and relative terms (i.e., in relation to other daily events) - with implications for how the scheduling of daily meals and nutrients can affect the acute and chronic regulation of metabolism and behaviour.

The projected rise in world population to 9.7 billion by 2050 will require an increased production of meat globally to meet human requirements for protein without compromising environmental and financial sustainability. Efficient increase in meat production will require an in-depth knowledge of muscle growth and development. The ability of sows to produce large numbers of piglets per litter coupled with a farrowing rate of an average of two litters per year makes the pig a highly efficient source of meat. In addition, the anatomical and physiological similarities between pig and humans makes the pig an excellent experimental model to study human physiology and disease. The availability of robust in vitro models of skeletal muscle from livestock species is critical for understanding muscle development and disease, and to make progress towards improving animal meat production. A current technical limitation in this regard is that stem/precursor cells routinely obtained from muscle are a mixed population with low proliferative potential and low differentiation efficiency in vitro thus limiting their use in large-scale studies. This thesis aimed to isolate, purify, and characterise myogenic cells from porcine skeletal muscle. It also aimed to develop and characterise an explant-based culture model for the isolation and maintenance in vitro of muscle stem cells from pigs. Finally, with the aim to provide proof-of-concept of the usefulness of the novel in vitro model, studies were carried out to investigate the effects of β-Klotho (KLB) and its ligand, fibroblast growth factor 21 (FGF21) on adipogenic differentiation of pig muscle-derived progenitor cells using both a siRNA mediated knockdown and CRISPR/Cas9-mediated knockout of KLB. Using fluorescent activated cell sorting (FACS), I isolated porcine myogenic cells based on the expression of CD146 and obtained CD45-/CD31-/CD146+ and CD45-/CD31-/CD146- as myogenic and non-myogenic cell fractions, respectively. Analysis by RT-qPCR revealed that CD45-/CD31-/CD146+ fraction was indeed enriched for myogenic cells showing high expression of muscle stem cell markers, PAX7 and CD56, and low expression of preadipocyte marker, PDGFRα which was highly expressed in the CD45-/CD31-/CD146- fraction. In addition, when placed in myogenic differentiation media the CD45-/CD31-/CD146+ cells were able to fuse to form mulinucleaded myotubes with increased expression of muscle markers MYH3 and MYOG. Interestingly, no myotubes were observed in the CD45-/CD31-/CD146- fraction. On the contrary, both CD45-/CD31-/CD146+ and CD45-/CD31-/CD146- fractions were able to accumulate lipids and differentiate into adipocytes when placed in adipiogenic media. However, CD45-/CD31-/CD146- displayed a higher adipogenic capacity with significantly increased levels of expression of fatty acid binding protein, FABP4 when compared to CD45-/CD31-/CD146+ fraction. To establish an explant-based in vitro model, muscle tissue fragments were seeded on matrigel coated cell culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Expression of lineage markers in MDPCs was determined with the use of RT-qPCR and flow cytometry. In addition, their ability to differentiate into myogenic and adipogenic lineages during long term culture was also tested followed by RT-qPCR analysis of relevant lineage markers. The results showed that MDPCs displayed long-term expansion in vitro, showing an average doubling time of 48 hours. The MDPCs also expressed key muscle stem cell markers, PAX 7, MYOD, MYF5 and CD56 in the early passages while the adipogenic cell markers CD105 and PDGFRα were highly expressed at the later passages. In addition, the MDPCS were able to efficiently form myotubes over several passages. Eventually, these cells lost their myogenic potential and acquired adipogenic potential following prolonged culturing. Finally, to demonstrate the usefulness of this in vitro model for functional molecular studies in porcine muscle, I investigated the role of fibroblast growth factor 21-βKlotho (FGF21-KLB) signalling in regulating adipogenic differentiation of porcine MDPCs using both gain of function and loss of function approaches. RT-qPCR and Immunocytochemistry analysis revealed that KLB expression was upregulated in differentiating porcine MDPCs synchronous to the formation of adipocytes and upregulation of the adipocyte markers, PPARy and FABP4. Stimulation of porcine MDPCs with FGF21 increased adipogenic differentiation while siRNA mediated knockdown and CRISPR/Cas9 mediated knockout of KLB inhibited adipogenesis by porcine MDPCs indicating that FGF21-KLB signalling is a regulator of adipogenesis in pig muscle. In conclusion, these results show that CD146 is a suitable marker for isolation of myogenic cells from pig skeletal muscle using FACS. Moreover, I describe a simple and efficient method to purify muscle stem cells from pig skeletal muscle tissue and characterise their growth and differentiation potentials. This, together with novel know-how on gene targeting using CRISPRs provides valuable information towards future research applications to understand pig skeletal muscle development and improve meat production. Moreover, the novel in vitro model I developed could have applications in the expanding field of cultured meat. Lastly, the methods developed in this thesis may be transferable to other species provided culture conditions are appropriately optimised.

The high prevalence of obesity worldwide has fuelled research to discover new treatments that tackle obesity safely and effectively. Over the last decades, we and others have considered that developing strategies focused on increasing organismal energy expenditure may help fight obesity and its associated comorbidities. For this purpose, our research focused on a gene called Thada. THADA encodes a 220 kDa protein that has been recently reported in Drosophila melanogaster to uncouple the heat dissipation of SERCA from its Ca2+ pumping action. We anticipated that this mechanism could contribute significantly to the energy metabolism in endotherms, such as mammals. We envisaged THADA could contribute to the muscle non-shivering thermogenesis-in addition to sarcolipin, another uncoupler of SERCA. Our working hypothesis was reinforced by the fact that mutations in THADA have been positively selected during the last 45-30K years in the Arctic populations, which the authors of the research and we interpret as a potential adaption to the extreme cold. In this thesis, I have performed in vitro and in vivo approaches and confirmed that the ablation of THADA caused increased ATP hydrolysis and Ca2+ transport efficiency of SERCA and affected cellular bioenergetics in primary skeletal muscle cells. Interestingly, phenotypic characterisation of the Thada KO mice has revealed that the genetic ablation of Thada causes an impairment in the maintenance of core body temperature in response to cold exposure. This effect can only be revealed under specific environmental conditions when the brown adipose tissue (BAT) is cancelled/neutralised. Our physiological data are supported by a robust transcriptomic signature where skeletal muscle, BAT and inguinal subcutaneous white adipose tissue of the Thada KO mice presented primary and compensatory fingerprints associated with the environmental temperatures tested. The findings of this thesis provide evidence of THADA's contribution to energy homeostasis in mammals and open the possibility of using THADA as a viable therapeutic target to increase basal metabolic rate and hence energy expenditure to tackle obesity.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative and multi-system disease characterised by the gradual degeneration and loss of upper and lower motor neurons with associated muscle weakness, atrophy, and paralysis. Numerous mechanisms such as dysfunctional mitochondria and oxidative stress; axonal transport; glutamate excitotoxity; protein homeostasis; and dysregulated RNA processing are suggested to be responsible for the prevailing pathology,including extracellular vesicle-mediatedpropagation involving the transport of toxic misfolded and/or aggregated proteins. Extracellular vesicles, especially small extracellular vesicles (exosomes), are secreted byprincipal cell types implicated in ALS, including motor neurons, astrocytes, microglia and,recently reported, skeletal muscles. To investigate the contribution of the skeletal musclesecretome to pathology, this thesis focuses on the functionality and uptake mechanismsof skeletal muscle-derived small extracellular vesicles (or MuVs). In this thesis, a protocol for the efficient isolation of MuVs from conditioned media of myotubes was developed and validated, yielding MuVs with canonical markers characteristic of small extracellular vesicles. Secondly, vesicles secreted by differentiated myotubes exhibited selective toxicity based on functional studies in the principal cells affected in ALS (motor neurons, astrocytes, and skeletal muscle). MuVs derived from ALS myotubes were specifically and selectively toxic to motor neurons and myotubes, unlike larger vesicles or ectosomes derived from ALS myotubes that exhibited no toxicity. Thirdly, MuVs uptake by myotubes requires actin polymerization and the involvement of endocytic pathways such as macropinocytosis and clathrin-mediated endocytosis and lipid rafts. In addition, interactions of the EV surface with the recipient cell surface appear necessary for docking and subsequent internalisation of MuVs. Altogether, this work refines our understanding of the potential role of MuVs in vesicle-mediated propagation and the spread of toxicity in ALS.

The overriding aim of this thesis was to optimise, scale and utilise an in vitro model of skeletal muscle in order to establish a hypertrophic loading regime of engineered muscle, thereafter, characterising the molecular mechanisms following mechanical load and application of this regime. Mechanical loading of skeletal muscle results in molecular and phenotypic adaptations typified by enhanced muscle size (hypertrophy). However, skeletal muscle loss (atrophy) as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and sarcopenia, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. Studies in humans are limited by the need for repeated muscle biopsy sampling, and studies in animals have numerous methodological and ethical limitations. In this investigation, skeletal muscle was tissue engineered utilising the murine cell line C2C12, which bears both structural and functional resemblance to native tissue and benefits from the advantages of conventional in vitro experiments. The preliminary investigation (Chapter 3) aimed to determine if mechanical loading would induce an anabolic hypertrophic response, akin to that described in vivo following mechanical loading in the form of resistance exercise. This data reported that mechanical loading (construct length increase of 15%) significantly increased Insulin-like growth factor-1 (IGF-1) and Matrix metalloproteinase-2 (MMP-2) mRNA expression 21 hours post overload, and levels of the atrophic gene muscle atrophy factor BOX (MAFbx) was significantly downregulated 45 hours post mechanical overload. In addition, downstream mammalian target of rapamycin (mTORC1) targets, ribosomal protein S6 kinase beta-1 (p70S6 kinase) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP-1) phosphorylation, were upregulated immediately following mechanical overload. Furthermore, maximal contractile force was augmented 45 hours post load with a 265% increase in force, alongside significant hypertrophy of the myotubes within the engineered muscle. Thus, overall, mechanical loading of tissue engineered skeletal muscle induced hypertrophy and improved force production. Chapter 4 aimed to determine the role of IGF-1 in skeletal muscle hypertrophy utilising a further developed model of mechanical load, allowing a higher throughput and more biomimetic model of skeletal muscle and mechanical stimulation. Data revealed that the IGF-1R inhibitor NVPAEW541 stunted IGF-1 mRNA expression and mTORC1 activation, alongside both myotube hypertrophy and increases in maximal force production temporally following mechanical load. This supports the notion that skeletal muscle hypertrophy in response to mechanical loading is primarily driven through the linear IGF-1-Akt-mTORC1 pathway. Furthermore, this study also demonstrates the application of this model system to study distinct molecular pathways underpinning skeletal muscle atrophy and hypertrophy. Moreover, the model offers potential avenues to peruse for future treatments for skeletal muscle atrophy, within an easily accessible and highly controlled system. Chapter 5 aimed to determine if mechanical load would offset dexamethasone (DEX) induced skeletal muscle atrophy in muscle engineered using the C2C12 murine cell line. Data revealed that mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX, regardless of whether the loading occurred before or after 24 hours of DEX treatment. This is likely to be underpinned by the prevention of upregulation of muscle RING-finger protein-1 (MuRF-1) and MAFbx mRNA expression, critical regulators of muscle atrophy. Therefore, I demonstrated the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy. The data presented in this thesis demonstrates the use of a novel mechanical stimulation bioreactor. Firstly, to investigate the beneficial therapeutic effects of mechanical load on atrophied engineered muscle, but also to further investigate the acute complex cellular and molecular responses. Specifically, this model has shed further light on the mechanoresponsive genes and proteins underpinning skeletal muscle hypertrophy following mechanical stimulation, offering potential therapies for skeletal muscle wasting. Future studies will investigate the use of this model to either feed forward or feedback on human exercise science experimentation to better inform what we already know around the area of exercise as a treatment for muscle wasting.

Physiological glucose levels are maintained by the complex integration of neuroendocrine, hormonal and nutritional signals controlled by multiple tissues in the body. A dysregulation in these mechanisms leads to increasingly prevalent conditions characterised by an inability to regulate blood glucose levels, such as diabetes. Maintaining glycaemia within a target range remains a daily challenge for individuals with both Type 1 and Type 2 diabetes and a better understanding of the pathophysiology of impaired glucose homeostasis in these conditions is still required to identify more effective and targeted therapeutic approaches. Work in this thesis focused on elucidating the mechanisms by which lipid overflow, be it from increasingly sedentary behaviour or overfeeding, leads to the development of insulin and anabolic resistance in skeletal muscle. Loss of insulin-stimulated glucose clearance by skeletal muscle is a main driver for impaired glucose disposal in Type 2 diabetes and a role for excessive lipid availability in this pathology is well established. Here, muscle cells were treated with high concentrations of a saturated fatty acid and data demonstrated that lipid overflow led to impaired anabolic sensitivity, inflammatory cytokine release and mitochondrial dysfunction. Furthermore, these experiments elucidated a novel role for adenosine tri-phosphate, acting as a signalling molecule, in the regulation of muscle glucose metabolism, identifying insulin and exercise mimetic roles of the nucleotide that could be therapeutically targetable. This work was translated into humans, where the effect of lipid overflow by high-fat overfeeding was assessed in an experimental model of inactivity-induced insulin and anabolic resistance. Data suggested that two days of disuse (by forearm immobilisation) were sufficient to cause substantial muscle insulin resistance. After 7 days, muscle strength was significantly reduced and anabolic resistance was evident due to decreased forearm balance of potent anabolic amino acids such as leucine. Contrary to the hypothesis, high-fat overfeeding did not accelerate or exacerbate these impairments, suggesting that removal of contraction represents a potent stimulus for loss of substrate demand by muscle, irrespective of energy balance. Insulin replacement therapy has been the cornerstone of treatment for Type 1 and advanced Type 2 diabetes for over 8 decades. A serious and inadvertent consequence of prolonged insulin therapy is the increased risk of hypoglycaemia. Hypoglycaemia can lead to impaired physiological defences against a decrease in blood glucose and loss of awareness of these changes. AMP-activated protein kinase activators, which are widely used (to target peripheral tissues) as anti-hyperglycaemic agents in Type 2 diabetes have demonstrated central effects that amplify the first defence against hypoglycaemia, or counterregulatory response. Data presented here demonstrated that peripheral administration of a brain permeable AMP-activated protein kinase activator amplified the counterregulatory response to hypoglycaemia by enhancing glucagon levels in healthy rats, without altering fasting blood glucose. This demonstrates important clinical implications for the pharmaceutical use of AMP-activated protein kinase activators as the central roles that regulate blood glucose may supersede the peripheral effects of these compounds, during hypoglycaemia. Work presented here highlights the complexity of the regulation of glycaemia and discusses the contribution of extracellular and intracellular nucleotides/nucleotide sensors to glucose homeostasis. It can be concluded from this work that strategies to manage or treat diabetes in future should consider the importance of tissue-specific or metabolic status specific actions of the targets of interest.

Skeletal muscle has a high capacity for self-regeneration, however, this is limited when there is a significant loss of tissue. The development of cell based constructs using biomaterials that can stimulate cell proliferation and differentiation, could support the body's natural systems during regeneration. Cellulose nanowhiskers (CNWs) are rod like nanomaterials, typically 5 to 20 nm in diameter, and can range from 100's of nanometres to several microns in length. The high aspect ratio of the CNWs could provide contact guidance suitable for directing cells of highly structural tissues, like skeletal muscle. This work explores the potential of multilayer substrates composed of alternating polyelectrolyte CNW and chitosan, with an oriented CNW top layer to promote the alignment and differentiation of myoblasts in to myotubes. Multilayer substrates were assembled through the dip coating of CNW and chitosan solutions on to glass coverslips. CNWs are produced through the partial acid hydrolysis of cellulose. The use of sulphuric acid results in a residual negative charge caused by the addition of sulphate ester groups on the whiskers. As a polyanion, CNWs can be layered with an oppositely charged polycation, chitosan, to form stable multilayer films. CNWs can then be spin coated on to the multilayer substrates forming a radial oriented nanotopography. Once formed, the capabilities of the substrates to promote cell growth and differentiation were investigated using C2C12s, an immortalised mouse myoblast cell line. The cells showed distinct signs of differentiation through the immunofluorescence staining of key myogenic components. Broad alignment of myotubes was observed on the oriented CNW multilayers whereas only local alignment formed on the substrates without the CNWs. Other cell types also showed a morphological cell response to these substrates, as human skeletal muscle cells (hSkMCs) and bone marrow derived mesenchymal stem cells (BM-MSCs) both showed alignment and elongation on the oriented CNWs. To further encourage the proliferation and differentiation of cells, key extracellular matrix (ECM) proteins, such as fibronectin and laminin, were adsorbed onto the substrates. Both proteins showed increased cell spreading and proliferation compared to untreated controls for C2C12 and BM-MSCs. These results are extremely promising for development of nanotopographical substrates, and pave the way for more complex 3D tissue scaffolds to be developed.

A variety of metabolic disease states as well as the ageing process are characterised with a state of low-gradechronic inflammation, which may contribute to skeletal muscle wasting. Since muscle wasting is associated with increases in morbidity and all-cause mortality, targeting of skeletal muscle inflammation may prove a useful therapeutic tool to enable the preservation of skeletal muscle mass in disease states categorised by chronic low-grade inflammation. One potential avenue for intervention is via nutritional intervention, omega-3fatty acids have been implicated to have anti-inflammatory properties mediated via a variety of mechanisms. One such mechanism is through a group of bioactive lipid mediators produced in inflammatory states via the metabolism of both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which aretermed specialised pro-resolving mediators (SPM's).

Approximately 340 million people worldwide have Type 2 Diabetes Mellitus (T2DM), making identification of the aetiological processes underlying this disease imperative. Endoplasmic reticulum (ER) stress has emerged as a potential mechanism driving the pathogenesis of obesity and T2DM. Palmitate, the predominant saturated fatty acid elevated in the blood plasma of obese individuals, induces ER stress and insulin resistance in skeletal muscle. However, the interaction between ER stress, lipid metabolism and T2DM remains poorly understood. This thesis uses lipidomic tools to investigate skeletal muscle ER stress in cell culture and animal models. Chronic palmitate treatment of both mouse C2C12 and human primary myotubes induced ER stress, concurrent with the cytosolic phospholipase A2-dependent release of polyunsaturated fatty acids (PUFAs) from phosphatidylcholines (PCs). This phenotype was also observed in skeletal muscle from mouse models of diet-induced obesity and T2DM patients. Palmitate-stimulated catabolism of PUFA-containing PCs was concomitant with increases in bioactive eicosanoid secretion, which was shown to be important in the control of ER stress, inflammatory signalling and macrophage activation. This provides a novel link between palmitate and the control of ER stress and inflammation in metabolic disease. Previous work has suggested that the propagation of ER stress signalling between cells may result from the secretion of a, as yet undefined, cell non-autonomous signal. In this thesis, long-chain ceramides (40:1 and 42:1) were identified as signals transmitting the induction of ER stress between myotubes via exosome-mediated transport. Long-chain ceramide concentrations were increased in skeletal muscle and blood plasma from in vivo models of obesity and were elevated in the muscle of T2DM patients. Muscle synthesis of long chain ceramides in response to palmitate was found to occur via the de novo pathway and was linked to ER stress by Perk, an important unfolded protein response kinase. This work identifies ceramides as cell non-autonomous signals that propagate ER stress activation following exposure to palmitate, providing a novel mechanism for stress signalling in obesity and insulin resistance.

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.

Skeletal muscle is a highly metabolically active tissue, both in the adult and the fetus. Mitochondria are essential in providing energy in the form of ATP from the oxidative metabolism of carbohydrates, fats and amino acids. Mitochondrial function is influenced by the abundance and activity of the complexes comprising the electron transfer system (ETS) and the balance between mitochondrial fusion and fission. Any factors which affect the development of skeletal muscle, and mitochondria in particular, may have an impact not only on neonatal health but also on the metabolic health of the adult offspring. However, the normal developmental profile of skeletal muscle mitochondrial function as the fetus prepares for the increased metabolic challenges associated with extrauterine life, is not well characterised. The hormones, cortisol and triiodothyronine (T3) are known to be crucial in the maturation of several physiological processes during late gestation. Further, their role in regulating adult metabolism is well-documented. However, whether they play a role in regulating fetal mitochondrial function is unknown. Using fetal sheep, the aims of this project were twofold: 1) to determine any changes in skeletal muscle mitochondrial function which occur over the last third of gestation and in the first two days of neonatal life and 2) to determine any regulatory roles of cortisol and T3 in these developmental changes. Mixed fibre-type skeletal muscle was collected from fetuses at 3 time points over late gestation and from newborn lambs. In addition, skeletal muscle samples were taken from fetuses which had been thyroidectomised (TX) and fetuses infused with either T3 or cortisol. Respirometry, enzyme assays, qRT-PCR and western blotting were carried out on the skeletal muscle samples in order to assess mitochondrial parameters. Mitochondrial activity, as measured by carbohydrate- and fat- stimulated ADP-coupled oxygen uptake, increased with age in a thyroid hormone dependent manner, rising predominantly postnatally. Mitochondrial density, abundance of ETS complexes I-IV and ATP-synthase and expression of the adenine nucleotide transferase 1 and mitofusin 2 were all positively influenced by age, with the natural prepartum rise being prevented in the thyroidectomised fetuses. However, T3 infusion alone was insufficient to raise any of these factors prematurely. Cortisol infusion resulted in an increase in some aspects of mitochondrial oxidative capacity in a muscle-specific manner. Overall, the data presented shows that there are developmental changes in skeletal muscle mitochondria during the perinatal period. They also suggest that these changes are regulated by both cortisol and thyroid hormones in preparation for birth, although neither hormone alone was sufficient to induce all the functional changes.

Genome Wide Association Studies (GWASs) have identified variants associated with disease that promise to deliver insights into disease aetiology. However, because many GWAS variants lie in non-coding genomic regions, it is difficult to define the genes and pathways underlying a GWAS signal. The possibility of linking GWAS variants to molecular traits, combined with the development of high throughput assays, has motivated the mapping of molecular quantitative trait loci (QTLs), genetic associations with molecular traits such as gene expression (eQTLs) and DNA methylation (mQTLs). The Finland-United States Investigation of NIDDM (FUSION) tissue biopsy study is motivated by the desire to understand the molecular pathogenesis of Type 2 diabetes (T2D), a complex disease where the vast majority of the ~100 independent GWAS loci occur in non-coding regions. To elucidate the molecular mechanisms underlying these signals, we collected skeletal muscle biopsies, a T2D-relevant tissue, from 318 extensively phenotyped individuals who exhibit a range of glucose tolerance levels. From these biopsies, we generated genotype, gene expression, and DNA methylation information, enabling us to directly measure the effects of T2D on molecular traits, and to link non-coding T2D GWAS loci to candidate molecular targets. In this thesis, I present a catalogue of genetic effects on gene expression and DNA methylation. I use this catalogue firstly, to reveal basic biology of the genetic regulators of skeletal muscle molecular traits, and secondly, to identify molecular traits that are relevant to T2D, glycemic, and other complex traits. In regards to basic biology, I characterise the broader genomic context of QTLs by calculating the enrichment of QTLs in chromatin states across a diverse panel of cell/tissue types. I also identify key skeletal muscle transcription factors (TFs) and classify them as activators or repressors by aggregating the effects of QTLs predicted to perturb TF binding sites. In addition, I characterise the properties of methylation sites associated with gene expression and use inference models to dissect these methylation-expression relationships, classifying cases where the genetic effect is mediated by methylation, expression, or is independent. I also integrate molecular trait genetics with complex traits. First, I perform a conditional analysis, mapping GWAS variants for T2D and glycemic traits to molecular traits, prioritising disease relevant skeletal muscle molecular traits. Second, recognising QTLs may also be specific to a disease state or environmental context, I leverage the rich phenotyping of participants to map genotype by environment (GxE) effects on gene expression—eQTLs that exhibit effects specific to an environmental context. Altogether, these analyses form a thorough survey of the genetic regulators of skeletal muscle expression and DNA methylation, and provide an important resource for interpreting complex diseases.

Toll-like receptor 4 (TLR4) activation increases pro-inflammatory cytokine production and upregulation of muscle atrophy signaling pathways. Exercise is recommended to reduce TLR4 expression and intracellular signaling, but the mechanisms for how this occurs are unknown, and even less is known regarding the effect of exercise on TLR4 expressed in skeletal muscle. Activation of TLR4 expressed on skeletal muscle can elicit local metabolic and atrophic effects in addition to systemic inflammation from TLR4 activation on immune cells. Therefore, the purpose of this dissertation project is to better uncover the nature and mechanisms by which exercise affects TLR4 expression and intracellular signaling in different cell types (peripheral blood mononuclear cells: PBMCs and skeletal muscle) using cell culture and human experiments. In this project, we showed that in an in vitro model of muscle contraction, repeated myotube contractions can prevent TLR4-mediated myotube atrophy, partially due to decreasing membrane-bound TLR4 and increasing soluble TLR4 (sTLR4). Next, we provided evidence that divergent mechanisms may regulate TLR4 expression on PBMCs by augmenting membrane-bound and soluble TLR4 expressions following workload-matched exercise in normoxic and hypoxic environments. Finally, we demonstrated that TLR4 receptor regulation and cellular signaling responses to vigorous exercise are dependent on training status (endurance-trained vs untrained) but not tissue type (PBMCs vs skeletal muscle). Together these studies provide a better understanding of TLR4 regulation in response to an acute bout of exercise in different tissue types and how endurance training may affect these responses.

Highly-trained athletes are accustomed to varied and high-volume based exercise stimuli and eliciting adaptation in individuals already possessing the necessary physiology to compete at the highest level is difficult. Therefore, identifying novel, potent and time efficient methods of achieving cumulative training stress is a continual quest for coaches and exercise scientists. This thesis examined the acute and chronic effects of manipulating exercise and recovery during brief all-out sprint cycling on adaptive responses favouring enhanced endurance capacity. Chapter 3 highlighted that low-volume non-work matched all-out sprint cycling, whether it be interval- (4 x 30 s bouts) or continuous based (1 x 2 min bout) provides a similarly potent stimulus for the acute induction of cell signalling pathways and key growth factors associated with mitochondrial biogenesis and angiogenesis in trained individuals. In line with manipulating recovery and in attempting to identify a novel and potent exercise intervention capable of giving athletes more return on their training investment, Chapters 4-6 investigated the efficacy of combining sprint interval training with post-exercise blood flow restriction (BFR). Firstly, it was demonstrated that BFR potentiates HIF-1α mRNA expression in response to SIT, tentatively suggesting an enhanced stimulus for hypoxia- and/or metabolic-mediated cell signalling associated with mitochondrial biogenesis and angiogenesis over SIT alone. Secondly, four weeks of SIT combined with post-exercise BFR provides a greater training stimulus over SIT alone in trained individuals to enhance VO2max (4.7 v 1.1 % change) and MAP (3.8 v 0.2 % change), but not 15-km TT performance. Finally, in response to four weeks of SIT combined with post-exercise BFR, an international female track sprint cyclist increased her CP and W by 7 and 2 % and VO2max and absolute MAP by 3 and 4 %, respectively. Through a combination of an acute in vivo molecular experiment, a training study and an athlete case study, this thesis has introduced a potentially potent and novel training concept that appears capable of augmenting aerobic capacity.

Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.

Assessments of skeletal muscle functional capacity or bilateral muscular asymmetry often necessitate maximal contractile effort, which exacerbates muscle fatigue or injury. Tensiomyography (TMG) has been investigated in laboratory settings, as a means to assess muscle contractile function following fatigue; however observations have not been contextualised by concurrent physiological measures. TMG has more sparingly been applied in the field, with elite athletes. The aim of this thesis was to examine acute alterations and underlying variations in muscle contractile mechanics, through the application of TMG, contextualised with established physiological measures; and to apply TMG within high performance sports programmes. TMG successfully detected fatigue, evident from reduced strength, by displaying impaired muscle displacement, accompanied by elevated resting muscle tension. Greater asymmetry was detected in individuals with asymmetric strength; however, symmetry was masked during more complex tasks. Increased day-to-day variability was detected among highly trained athletes compared to recreationally active individuals. Acute training adaptations were detected, in contractile mechanics, in individual muscles. TMG could be useful in establishing fatigue status of skeletal muscle without exacerbating the functional decrements of the muscle, whilst also providing useful screening information for detecting asymmetry which may not be apparent during functional actions.

Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 μCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism.

During skeletal muscle contraction, glucose is imported into the muscles cells to fuel contraction and Sodium Potassium-ATPase (NKA) is also activated to maintain the sodium (Na) and potassium (K) gradients needed for the excitation-contraction coupling process. It is well established in other tissues, that many nutrient transporters such as the Sodium-Glucose Linked Transporters (SGLTs) use the Na gradient established by NKA to import Na ions and glucose into a cell. Insulin-Independent glucose uptake has been known to exist in contracting skeletal muscles since the mid-1980s. However, despite 30+ years of research, a mechanism has remained elusive. If a SGLT were present in skeletal muscle it would represent an insulin-independent glucose uptake mechanism.NKA coupled to an SGLT has not been investigated in skeletal muscle mainly due to a lack of methods to measure co-transported species. K ions and glucose are exchanged by NKA and an SGLT respectively, therefore both species would have to be simultaneously measured. To investigate whether this insulin-independent process operates in skeletal muscle, we developed a novel method to measure simultaneously non-radioactive 13C-labeled glucose and rubidium (85Rb) in biological samples using Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS). Rb is used as a congener for K as NKA pumps 3 Na ions out of the cell and imports 2 K (or Rb) ions into the cell. Rb uptake can therefore be used to measure the activity of the NKA pump. Using our novel method, we discovered a previously unrecognized glucose uptake mechanism in skeletal muscle. In this mechanism, a SGLT imports glucose together with Na ions, driven by the contraction stimulated activity of NKA α2. We measured simultaneous glucose and NKA transport in isolated mouse Extensor Digitorum Longus muscles using ICP-MS with 13C-substituted glucose substrates and Rb. The SGLT family transports alpha-methyl-D-glucoside (αMDG), a substrate not handled by GLUT family transporters, and is inhibited by ouabain at concentrations that selectively inhibit the NKA-α2 subunit isoform. It can be activated independently by muscle contraction or pharmacological stimulation of NKA. SGLT uptake is additive with GLUT-mediated uptake during and post contraction. The uptake rate of SGLT varied over a wide range, suggesting that it is regulated by contractile or metabolic conditions that remain to be defined. Consistent with the observed functional uptake, we detected the SGLT2 gene SLC5A mRNA transcripts by RT-qPCR, and SGLT2 protein by immunohistochemistry. However, the possibility that this SGLT transporter is a muscle specific isoform of SGLT2 that is distinct from kidney cannot be eliminated. These results establish SGLT2, or a related variant, as an important transporter in insulin-independent glucose uptake by skeletal muscle. In addition, they establish ICP-MS with 13C-substituted nutrients as a useful technology for studies of coupled nutrient transport in other biological systems. A more complete understanding of the molecular mechanisms of insulin-independent glucose uptake in muscle may provide further evidence of why exercise is such a cornerstone therapy for diabetes when muscles become insulin resistant. This in turn may provide a new therapeutic target for treating diabetes.

Perfluorooctanesulfonate (PFOS) is a persistent environmental pollutant previously used in industrial products such as fire-fighting foams, furniture, and stain-resistant coatings. PFOS is associated with adverse health outcomes, including hepatosteatosis and diabetes. Previously, humanized CYP2B6-transgenic (hCYP2B6-Tg) mice accumulated more PFOS than Cyp2b-null mice and experienced greater mortality; however, the Cyp2b-null mice experienced greater steatosis. Cyp2b-null and hCYP2B6-Tg mice were treated with 0, 1, or 10 mg/kg/day PFOS for 21 days. To determine how PFOS affected the metabolism of polyunsaturated fatty acids (PUFAs) oxylipin concentrations were measured via LC-MS/MS. Additionally, gene expression changes were measured using RNA sequencing. PFOS treatment increased the expression of soluble epoxide hydrolase enzymes which correlated with increased concentrations of diol- and decreased epoxide-containing oxylipins. PFOS-mediated liver toxicity was also associated with perturbed arachidonic acid metabolism. Several hepatic Oatps (Oatp1a4-6) were increased in the hCYP2B6-Tg females compared to the Cyp2b-null females, which may have increased hepatic PFOS uptake and retention. In addition to altered hepatic lipid metabolism, PFOS-treated Cyp2b-null and hCYP2B6-Tg mice experienced muscle wasting with PFOS. Our lab has previously demonstrated reduced mitochondrial capacity in PFOS-treated C2C12 cells, so we wanted to determine if mitochondrial function was affected in the Cyp2b-null and hCYP2B6-Tg skeletal muscle. We performed Respirometry in Frozen Samples (RIFS) assay to measure mitochondrial complex function. PFOS decreased complex I and IV function. The purpose of this study is to elucidate PFOS’ mechanism of hepatic retention, characterize how it changes PUFA metabolism and its effect on skeletal muscle metabolism.

The time course of this increase in muscle n-3 PUFA composition and anabolic protein expression is currently unknown. In Chapter 2 of this thesis ten healthy male participants consumed 5g.d-1 of n-3 PUFA-enriched fish oil for 4 weeks. Muscle biopsies samples were collected in the fasted, rested state 2 weeks prior, immediately before (Week 0), at Week 1, Week 2 and Week 4 after initiation of fish oil supplementation for assessment of changes in lipid composition and expression of anabolic signalling proteins over time. Muscle lipid profile, (% total n-3 PUFA/total fatty acids) increased from W0 to W2 (3.8 ± 0.2 to 5.1 ± 0.3 %) and continued to rise at W4 (6.7 ± 0.4 %). Total protein content of FAK increased from W0 to W4 (3.9 ± 1.5 fold) whereas total mTOR was increased from W0 at W1 (2.4 ± 0.6 fold) with no further significant increases at W2 and W4. For the first time this study demonstrates that oral fish oil consumption results in an increase of n-3 PUFA levels in human skeletal muscle that is associated with increases in the expression of anabolic signalling proteins. Our understanding of the anabolic signalling process that underpins muscle protein synthesis has been advanced by the application of the WB technique. However, the semi-quantitative nature and poor dynamic range associated with the WB technique may lead to incongruence regarding the molecular response of skeletal muscle to anabolic stimulation. Chapter 3 of this thesis developed and applied a quantitative in vitro [γ-32P] ATP kinase assay (KA) alongside a traditional WB methodology to assess p70S6K1 signalling responses in human skeletal muscle to RE and protein feeding. Following validation in tissue culture with rapamycin and optimization of the assay in human skeletal muscle, this methodology was tested in a physiologically relevant context. In this regard, six males performed unilateral resistance exercise (RE) followed by the consumption of 20 g of protein. Skeletal muscle biopsies were obtained at pre-RE, at 1 h and 3 h post-RE. In response to RE and protein consumption, p70S6K1 activity was significantly increased from pre-RE at 1 h and 3 h post-RE (8.84 ± 0.78 to 17.18 ± 2.62 and 15.62 ± 3.12 µU/mg). However, phosphorylated p70S6K1thr389 was not significantly elevated. To assess if a combined stimulus of RE and feeding can influence AMPK activity we directly measured AMPK activity. AMPK activity was suppressed from pre-RE at 3 h post-RE (24.15 ± 1.6 to 15.64 ± 1.07 mU/mg), whereas phosphorylated ACCser79 was unchanged. These data therefore highlight the utility of the KA to study skeletal muscle plasticity. Previous studies have shown that ingestion of n-3 PUFA potentiates the phosphorylation of mTORC1 and associated kinases in response to nutrition. However, no study has identified whether n-3 PUFA supplementation potentiates anabolic kinase activity when RE is performed prior to nutrient provision. In Chapter 4 of this thesis, twenty healthy males consumed 5g.d-1 of either fish oil (FO) or coconut oil (CO) capsules for 8 weeks. Muscle biopsy samples were collected in the fasted, rested state before and after 8 weeks of supplementation for assessment of changes in lipid composition. Following 8 weeks of supplementation muscle samples also were obtained at rest (Rest), post RE in both the exercise leg (Post-RE) and the rested leg (Pre-FED) and also at 3 h post RE and protein feeding from both the exercise leg (3 h post-REF) and rested leg (3 h post-FED). There was a 2-fold increase in muscle (5.53 ± 0.3 to 11.16 ± 0.45 % of total fatty acids) n-3 PUFA composition after supplementation in the FO group but no change in the CO group. Following supplementation there was an increase in p70S6K1 activity at 3 h post-REF from Rest in the CO group (5.6 ± 1.4 to 12.2 ± 2.1 µU/mg) but no change in the FO group. In the CO group, AMPKα2 was significantly increased at Post-RE from Rest (3.7 ± 0.7 to 9.9 ± 2.0 mU/mg). These data show that 8 weeks of n-3 PUFA enriched fish oil supplementation suppresses the activity of p70S6K1 in response to RE and protein feeding.

The aim of this Thesis was to further characterise and utilise an in vitro skeletal muscle (SkM) model, to investigate its potential use in further understanding the cellular and molecular adaptations to exercise in vivo. Candidate genes and proteins have been identified using in vivo, ex vivo and targeted in vitro experiments, however the complete picture of these molecular mechanisms are far from understood. Furthermore, the extent to which mechanical signals contribute to the intra-cellular mechanisms associated with exercise is also underinvesitgated. To this end, developing an in vitro model of SkM that can recapitulate in vivo SkM and respond to mechanical stimulation in a similar way to exercise will provide a means to begin to delineate the complex cellular and molecular regulation of SkM. The initial investigation (Chapter 3) characterised an optimal seeding density and culture period of C2C12 myoblasts within a 3 ml collagen gel. These data provided support for the use of collagen constructs seeded at 4 x 106 cells/ml, with no statistical differences observed in peak force, rate of force development and relative force compared to other seeding densities examined (table 3-2, all p > 0.05). However the use of 4 x 106 cells/ml supports previous data in a larger construct volume model, whilst the highest cell density possible in the system increases cell-cell contact required for fusion. Immunohistochemical and gene expression analyses provided evidence for the fusion of single seeded myoblasts into multinucleate myotubes, demonstrating an in vivo-like architecture. Chapter 4 presented data towards the characterisation and use of two distinct cyclical stretch regimens with respect to the acute biochemical and transcriptional responses. Data revealed increases in peak media lactate and reductions in peak media glucose, following cyclical stetch compared to control (p = 0.000 and p = 0.001 respectively, Fig. 4-2). Changes in mtDNA (Fig. 4-5) and associated mRNA transcriptional signals (Fig. 4-7) were mode dependent.

Cellulose nanowhiskers (CNWs) are high aspect ratio rod-like nanoparticles with diameters on the order of a few nanometers. For the very first time CNWs are demonstrated as a useful material for guided tissue engineering. Due to their nanoscale dimensions and high aspect ratio, highly oriented spin coated surfaces of CNWs are shown to direct the morphology and terminal differentiation of myoblasts, allowing the culture of skeletal muscle-like tissue with a more physiologically relevant structure.CNWs are prepared from cellulose extracted from the tunicate Ascidiella sp. using acid hydrolysis to prepare high aspect ratio particles with diameters of approximately 5 to 6 nm. A spin coating method is used to prepare sparsely adsorbed sub-monolayers of CNWs with a high degree of relative orientation. The surfaces have a mean feature height of only 5.5 nm and the degree of CNW adsorption and orientation is modulated by altering the preparation procedure. When C2C12 myoblasts are seeded to the surfaces, the cells adopt highly oriented morphologies induced by the CNWs via contact guidance. This is a demonstration of contact guidance on some of the smallest topographical features ever reported. Furthermore, the highly oriented CNWs promote fusion and terminal differentiation of the myoblasts to form multinucleated myotubes with a striking degree of parallel orientation.CNW surfaces are also shown to support the adhesion and spreading of human mesenchymal stem cells, inducing the adoption of highly oriented cell morphologies. The ability of hMSCs to undergo cell fusion with C2C12 myotubes highlights the great potential for tissue engineering human skeletal muscle, using CNWs to direct the structure of the tissue. The bioactivity and low cytotoxicity of CNWs, coupled with their low cost and simple production procedure, indicates that CNWs will be a useful material for tissue engineering and regenerative medicine.

Skeletal muscles are composed of thousands of muscle fibres (muscle cells), densely packed together in parallel and surrounded by connective tissue sheaths. These fibres are multinuclear in nature, which allows for the control and regulation of the highly organised, protein rich cellular interior. The primary function of skeletal muscle is to produce force, which allows for movement to occur or posture to be maintained, and the regulation of this function is in turn reliant on the expression of specific genes and proteins. Skeletal muscle exhibits a high degree of plasticity, and can adapt in response to stimuli such as increased/decreased use, metabolic perturbations or changes in the systemic environment which often occur as a result of exercise, ageing, disuse or disease. Examining responses and adaptations in skeletal muscle in vivo are challenging due to experimental restrictions, and studies are limited by ethical issues surrounding experimentation on human beings and indeed on animals following the principals of refinement, reduction and replacement. Thus in vitro studies are often conducted in order to further understand mechanisms involved in adaptation. However, the environment to which skeletal muscle cells are exposed to in vitro is far removed from that in the body, and the resulting cellular architecture is often abnormal in morphology. Tissue engineered skeletal muscle has shown much promise in rectifying these issues, as cells can be grown on/within a matrix which is biologically relevant and engineered to grow in a uniaxial manner in parallel to one another. However, this field is in its relative infancy, and to date little data exists with regards the behaviour and characteristics of human muscle derived cells (MDCs) in tissue engineered constructs. In this thesis, human skeletal MDCs were obtained, characterised and subsequently cultured in a suitable model for tissue engineering purposes. MDCs were seeded on to a fibrin based hydrogel, which self-assembled over time to form a cylindrically shaped construct held in place between two anchor points. In ii this model, the cells were shown to align uniaxially and in parallel to one another in a fascicular like structure. The model was improved in terms of biomimicity and maturation by both increasing the seeding density of the MDCs, and by increasing the ratio of myogenic to non-myogenic cells. These models appear to promote the development of a slow muscle, as evidenced by the favourably high levels of MYH7 transcription in comparison to other isoforms, and showed suggestions of sarcomeric organisation as indicated by the classically striated pattern of protein organisation when myosin heavy chain immunostaining was conducted. The work conducted in the final chapter of this thesis focussed on developing a system capable of assessing and quantifying the force produced by these tissue engineered human skeletal muscle constructs when electrically stimulated. Further work in this area should aim to determine these functional characteristics and thereafter use the model for physiological, cellular and molecular studies in exercise science.

The aim of this project was to create a novel biodegradable, synthetic scaffold that will provide the correct topographical cues for myoblast alignment and efficient differentiation into myotubes. Skeletal muscle repair after major surgery or serious burns is often overlooked leading to poor healing and consequent loss of power in movements of affected limbs. In order to overcome this problem a tissue engineered construct could be utilised as a grafting patch to encourage further regeneration and enhance possible power to the limb. Using a biodegradable polymer can provide structural support until the tissue is established, and will be excreted by the body's natural processes as it degrades. A synthetic polymer is desirable as it can reduce the risk of immunogenic responses thus reduce risk of graft rejection. For successful in vitro growth of skeletal muscle, the cells must be encouraged to arrange themselves into parallel arrays in order for efficient fusion and consequent contraction. By incorporating the correct topographical cues into the scaffold to promote contact guidance for cellular alignment this can be achieved. Electrospinning is a reliable technique which yields highly reproducible aligned fibres from the micro- to the nanoscale. This project focuses upon creating and characterising the electrospun scaffold, checking biocompatibility with myoblasts by monitoring the topography, residual solvent within the scaffold, the mechanical properties of the scaffold, and a brief investigation into the degradation profile of the electrospun fibres. The immunogenicity of the scaffold was investigated by monitoring cytokine release from macrophages. Myoblast morphology was monitored, as was the efficiency of the cells to differentiate and their potential to become contractile myofibres. Cellular adhesion to the scaffold was also looked into by measuring the expression of integrins during early and late adhesion and on substrates with different topographies. It was found that the electrospun scaffold did not contain a significant amount of residual solvent, and macrophages were not activated any more than on tissue culture plastic. Myoblasts responded to the topography of the aligned fibres by aligning along the length of the fibres, showing elongation and bi-axial cytoskeletal arrangement after just 30 minutes culture on the aligned fibres. This elongation prompted fusion and differentiation of the myoblasts to occur faster than cells which were not exposed to the aligned topography, and this global alignment was maintained in long term culture.

Myotonia Congenita (MC) is a rare, inherited ion channelopathy caused by a loss-of-function mutation in the CLCN1 gene. The resulting downregulation of the skeletal muscle chloride channel (ClC-1) results in hyperexcitable skeletal muscle fibers that fire action potentials involuntarily. Patients with MC suffer from debilitating stiffness due to myotonia, described clinically as delayed muscle relaxation following voluntary contraction. Skeletal muscle is a unique system we use in this study to advance our understanding of the pathophysiology underlying MC, and other channelopathies characterized by hyperexcitable cells (i.e., forms of epilepsy and cardiac arrhythmia). Furthermore, re-assessing what makes anti-myotonic drugs such as mexiletine efficacious, yet imperfect, may help us improve the quality of life of affected patients through optimizing treatment outcomes.The current therapeutic approach to treating myotonia is to block voltage-gated Na+ channels in skeletal muscle (Nav1.4) to reduce the fast-transient Na+ current responsible for action potential generation (NaT) via use-dependent block (Statland et al., 2012),(Lo Monaco et al., 2015). Two assumptions are made by the field when taking this approach. First, open channel blockers, those that increase their block of Na channels during repetitive firing, are the preferred type of drug. Second, the block of NaT is the mechanism underlying efficacy. We tested both hypotheses utilizing both transgenic and pharmacologic models of MC in mice. Techniques used included current clamp of the extensor digitorum longus (EDL) muscle and voltage clamp of the flexor digitorum brevis (FDB) muscle. We compared the efficacy of two open-state NaCh blockers; mexiletine and ranolazine, to a closed-state blocker, µ-conotoxin GIIIA (uCTX). We quantitated efficacy against myotonia and determined the optimal concentration of each drug using intracellular recordings from EDL fibers. We were surprised to find that open-channel blockers were not more effective than the closed-channel blocker, uCTX. Using voltage clamp we quantified the reduction of both NaT and NaP caused by each drug in individual, dissociated muscle fibers from the flexor digitorum brevis (FDB). We found block of NaT does not correlate with the elimination of myotonia. In contrast, block of NaP (a non-inactivating, persistent Nav1.4 current) correlates well. In collaboration with Dr. Brent Foy, we performed a computer simulation of myotonia and found block of NaP, rather than NaT, appears to be the mechanism underlying efficacy. This work suggests a paradigm shift for the field in which selective targeting of NaP is the goal. A corollary is that block of NaT is an unwanted side effect of therapy. We studied whether findings in paralyzed single muscle fibers were predictive of the response of contracting whole EDL muscles to treatment with Na channel blockers. We were surprised to find that uCTX was significantly less effective compared to ranolazine and mexiletine in contracting muscles in the pharmacologic model of MC. The difference was significantly less in the genetic model of MC. These data are consistent with the possibility that both ranolazine and mexiletine block a second contributor to myotonia. That contributor appears to be downregulated in the genetic model of MC, in which myotonia is present chronically. We combined treatment with uCTX with a blocker of TRPV4, a stretch-activated channel, and found a modest improvement in efficacy. Efforts are ongoing to determine the additional channel type(s) blocked by mexiletine and ranolazine.Our data suggest 3 conclusions; (1) Block of NaP rather than NaT is the mechanism underlying efficacy of Na channel block in lessening myotonia, (2) Open channel block is not a superior approach to therapy. (3) A stretch-activated channel, which is blocked by ranolazine and mexiletine, is an important contributor to the generation of myotonia in contracting muscle.

Stimulated contractions are shown to increase toll-like receptor 4 (TLR4) release from C2C12 myotubes in vitro. The purpose of this study was to determine if the release of TLR4 from myotubes is controlled by proteolytic membrane cleavage. A broad-spectrum metalloprotease inhibitor was used to test this hypothesis in a C2C12 cell culture model, and TLR4 concentrations in the cell culture supernatant were measured via enzyme-linked immunosorbent assay (ELISA) following stimulated myotube contractions. ELISA analysis revealed TLR4 concentrations below the sensitivity threshold of the ELISA kit for all experimental conditions. In confirming the effectiveness of the electrical pulse stimulation (EPS) treatment, ANOVA analysis showed myotube diameter was not significantly different between conditions (p > 0.05), and supernatant IL-6 concentrations were not increased with EPS treatment. These results reflect an abnormal response of the C2C12 myotubes to stimulated contractions here, and additional troubleshooting is needed to test the proposed hypothesis.

The effects of pH, Mg²⁺ and Ca²⁺ upon both the intrinsic tryptophan fluorescence of the Ca²⁺-ATPase, and the fluorescence of the Ca²⁺-ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl), were used to study the binding of Ca²⁺ to the Ca²⁺-ATPase. The tryptophan fluorescence of the Ca²⁺-ATPase was sensitive to the calcium occupancy of the high affinity Ca²⁺ binding sites of the Ca²⁺-ATPase and it is proposed that this is due to a conformational change occurring between binding of the first and second Ca²⁺ ions. The fluorescence of NBD-Ca²⁺-ATPase was shown to be sensitive to pH, Mg²⁺ and Ca²⁺ and is proposed to be sensitive to the E2-E1 transition. Computer modelling of the effects of pH, Mg²⁺ and Ca²⁺ upon both the intrinsic tryptophan fluorescence of the Ca²⁺-ATPase, and the fluorescence of NBD-Ca²⁺-ATPase at equilibrium, together with simulations of stop-flow experimental data has allowed the development of a comprehensive kinetic model for Ca²⁺ binding to the Ca²⁺-ATPase, and the effects of pH, Mg²⁺ and Ca²⁺ concentration on this binding. The use of the fluorescent nucleotide analogue 2'3'-0-(2,4,6-trinitrophenyl)-adenosine dephosphate (TNP-ADP), and the effect of ATP on the fluorescence of NBD-Ca²⁺-ATPase has allowed the model developed above to be expanded to include ATP binding.

One exercise session can increase insulin-stimulated glucose uptake (ISGU) in skeletal muscle. This effect can be sustained for many hours postexercise (PEX), but PEX carbohydrate refeeding can speed the reversal of exercise-enhanced ISGU. Muscle glycogen accumulation is often assumed to cause the reversal of exercise-enhanced ISGU. However, exercise and refeeding affect many processes, making it difficult to isolate the role of glycogen. The main purpose of this dissertation was to test the role of muscle glycogen in reversal of exercise-enhanced ISGU using a novel genetic model that relied on intramuscular injection of adeno-associated-virus (AAV) that delivers small hairpin RNA (shRNA) targeting glycogen synthase 1 (GS1; AAV-shRNA-GS1), and AAV-shRNA-Scrambled (AAV-shRNA-Scr) as a control. AAV-shRNA-GS1 was injected into one epitrochlearis muscle; the contralateral muscle was injected with AAV-shRNA-Scr in each rat. Study 1 tested the effectiveness of the genetic model for attenuating muscle glycogen accumulation in rats that were refed postexercise. Study 1 also evaluated the relationship between glycogen and postexercise effects on the abundance of key proteins and phosphorylation of AMP-activated protein kinase (pAMPK). Rats completed a swim exercise protocol (2-hours duration) or remained sedentary. AAV-treated muscles were dissected immediately postexercise (IPEX), 5-hours postexercise (5hPEX), or 9-hours postexercise (9hPEX) with time-matched sedentary controls. Rats from 5hPEX and 9hPEX groups with time-matched sedentary controls were refed (RF) or not refed (NRF). Muscles were analyzed for glycogen, abundance of multiple proteins (including GS1, hexokinase II, HKII; glucose transporter 4, GLUT4), and pAMPK. AAV-shRNA-GS1 versus paired AAV-shRNA-Scr muscles had substantially lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK. Neither of these IPEX-values differed for shRNA-GS1 versus shRNA-Scr muscles. Glycogen in RF-rats was lower for shRNA-GS1 versus shRNA-Scr muscles at 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed, regardless of GS1-knockdown or diet, and despite differing glycogen levels. These results: 1) established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects; and 2) revealed that exercise-effects on abundance of the proteins assessed did not correspond to differences in PEX glycogen. Study 2 used the genetic model validated in Study 1. Rats performed exercise or remained sedentary. Epitrochlearis muscles were dissected at 3-hours postexercise (3hPEX) or 6-hours postexercise (6hPEX) with time-matched sedentary controls. Rats were either RF or NRF. Isolated muscles were incubated in [3H]-3-Methylglucose with or without insulin. Muscles were analyzed for glucose uptake, glycogen concentration, abundance or phosphorylation of proteins, and AMPK-γ3 activity. Study 2 revealed: 1) GS1 abundance was lower for shRNA-GS1 versus shRNA-Scr muscles regardless of refeeding status, exercise status, and dissection timing; 2) in RF-3hPEX and RF-6hPEX rats, muscle glycogen was substantially lower for shRNA-GS1 versus shRNA-Scr muscles; 3) in NRF-rats, ISGU at 3hPEX and 6hPEX exceeded sedentary controls; 4) exercise-enhanced ISGU was not reversed with refeeding at 3hPEX with or without GS1-knockdown; 5) in spite of substantially lower glycogen for shRNA-GS1 versus shRNA-Scr, exercise-enhanced ISGU was reversed in each of the paired muscles at 6hPEX when rats were RF; 6) exercise increased AMPK-γ3 activity at 3hPEX, but with refeeding, exercise-effects on AMPK-γ3 activity were eliminated. Overall, the results: 1) validated a genetic model to reduce GS1 abundance and glycogen concentration in rat skeletal muscle; and 2) provided novel evidence that attaining a high muscle glycogen concentration did not directly cause RF-linked reversal of exercise-enhanced ISGU.

To date, the mechanisms that underlie skeletal muscle decline due to ageing remain unclear. A hypothesis known as the mitochondrial theory of ageing postulates that a loss of mitochondrial quality control (MQC) leads to an increase in mitochondrial dysfunction over lifespan. Consequently, this loss of MQC underpins the deterioration in quality and function of all tissues throughout the organism, including skeletal muscle. Aerobic exercise has been found to potently stimulate increases in mitochondrial function and has been found to also greatly benefit aged skeletal muscle quality and function, suggesting that exercise may be a powerful tool to mitigate aged related skeletal muscle mitochondrial decline. A primary aspect of mitochondrial quality control is known as mitophagy, which is defined as the selective removal and degradation of damaged or dysfunctional mitochondria from the mitochondrial reticulum. The inherently dynamic and transient nature of mitophagy has provided a significant methodological challenge to mitophagy research, with many previous studies having to rely upon semi-quantitative techniques as the sole measurement. Additionally, many studies have relied upon toxic mitochondrial uncoupler chemical agents to induce mitophagy which limit the investigative potential of such experiments. These difficulties have led to conflicting evidence as to how skeletal muscle mitophagy alters in the context of ageing, with no clear consensuses being reached to date. To overcome these limitations, the Mito-QC mitophagy reporter mouse model was developed to provide a robust and functional method to visualize and measure mitophagy in vivo. Therefore, the aim of this thesis was to employ the Mito-QC model to gain deeper insight into the mechanistic properties of skeletal muscle mitophagy under basal and exercised conditions, and to conclusively determine how mitophagy is altered due to the process of ageing. Under basal physiological conditions, it was found that there is a directly proportional relationship between oxidative capacity and the amount of mitophagy that occurs in young mito-QC mice. In the context of exercise, previous studies have provided evidence to suggest that aerobic exercise may induce mitophagy, which would provide a natural and safe method to investigate mitophagy, alleviating the need for toxic pharmacological agents. Using an acute exercise protocol, I determined that mitophagy is induced following a single bout of exercise in both male and female mito-QC mice. This finding demonstrates that aerobic exercise is capable of being a potent tool to investigate mitophagy. Additionally, through the use of knockout mito-QC mouse models, I also demonstrated that exercise induced mitophagy does not occur in the absence of key mitophagy enzymes parkin and PINK1. This study provided key insights into the mechanistic aspects of exercise induced mitophagy and suggests essential roles for parkin and PINK1. Studies in the context of ageing and skeletal muscle mitophagy have provided conflicting evidence. To determine how mitophagy changes due to ageing, I examined mito-QC skeletal muscle across a wide range of age groups from young mice (3months of age), middle aged mice (12 months), to aged (24 months), and observed no change in mitophagy due to ageing. However, at 26 months of age, skeletal muscle mitophagy was greatly elevated, which coincided with a significant decline in mitochondrial function. Thus, mitochondrial quality is speculated to begin deteriorating due to ageing at 26 months in mito-QC mice, leading to an elevation in mitophagy to remove the increased number of dysfunctional mitochondria. Interestingly, this was not observed in 26-month-old mice who underwent an 8-week endurance exercise protocol, which also resulted in an increase in mitochondrial function. This demonstrates a potent protective effect of aerobic exercise for maintaining mitochondrial quality and function in aged skeletal muscle. In summary, this thesis contributes novel insights into the mechanistic aspects of skeletal muscle mitophagy in the context of both aerobic exercise and ageing.

Skeletal muscle is a heterogeneous tissue comprised of diverse cell types and is highly adaptable and sensitive to stimuli, such as exercise. Epigenetics is the study of mechanisms that impact chromosomal regions to alter gene activity without an underlying change in the sequence of Deoxyribonucleic acid (DNA). One of the most widely studied epigenetic mechanisms is DNA methylation (DNAm), which plays an important role in perpetuating cell identity. To date, studies have shown that exercise causes widespread DNAm changes in skeletal muscle. However, these studies have remained at the whole tissue-level. This approach limits interpretation of results due to, 1) the influence of cell identities on DNAm, 2) the variable cellular composition of tissue and 3) the specific responses of each cell type to exercise. This thesis investigates the genome-wide DNAm profiles of Type I, Type IIa and Type IIx multi-nucleated muscle fibres from human muscle at baseline and after twelve weeks of High Intensity Interval Training (HIIT). It describes the development of a novel method for the simultaneous extraction of DNA and protein from pooled human skeletal muscle fibre fragments (Chapter 4). The thesis also compares the methylome and proteome assessed in skeletal muscle fibre types using the same biological samples (Chapter 5 & 6), and in response to twelve weeks of HIIT (Chapter 7). This research will contribute to a deeper understanding of fibre-type specific muscle biology and molecular adaptations to exercise, with potential to improve treatment strategies for muscle related diseases and maintenance of muscle health.

Complex traits are intricately intertwined with an organism's genome, a relationship underscored by the dynamic landscape of its transcriptome. Selective gene expression regulates cell type specialization and fluctuation of cell states. The development of RNA sequencing has facilitated the capture of the whole transcriptome of a given sample. However, a bulk approach obscures cell type heterogeneity, impeding the precise dissection of cell-specific effects, including those modulated by genotype, developmental stage, and disease state. In contrast, single-cell and single-nucleus RNA-seq preserves cellular identity, enabling a comprehensive mapping of gene expression across various cell types and states.Here, I describe my work in single-cell transcriptomics to characterize cell types and cell states in mouse. First, I present our long-read single-cell RNA-seq method, benchmarked in the C2C12 mouse myogenic system, which revealed cell type-specific isoform switching in key genes during myogenesis. Next, I characterize 5 mouse tissues at single-nucleus resolution during postnatal development using the ENCODE4 mouse dataset, where I used topic modeling to reveal cell type- and state-specific cellular programs. Lastly, I investigate the impact of genetic variation on gene expression across 8 diverse tissues from 8 mouse genotypes, pinpointing genotype- driven variation in specific celltypes in both wild-derived and classical lab strains. Together, these projects lay the groundwork for cohesive cell type and cell state annotation and comparative analyses, contributing to future characterization of these tissues in other contexts such as human diseases and hybrid mouse genotypes.

Autophagy/mitophagy are cellular processes that play pivotal roles in maintaining mitochondrial function; thus, understanding their responses to high-intensity interval exercise (HIIE) can provide insights into the overall adaptive mechanisms of skeletal muscle. This study aimed to investigate the effects of a single session of HIIE with and without hypoxia on transcriptomic response relating to autophagy/mitophagy. Ten healthy males (age: 26 ± 4 years; BMI: 23.3 ± 2) participated in three HIIE sessions randomly order: hypoxia (HY, simulated altitude of 3200m with an oxygen fraction of 0.14), normoxia matched to the relative intensity of hypoxia (NR), and normoxia matched to the absolute intensity of hypoxia (NA). Skeletal muscle samples were collected at fourtime points: before HIIE (B), immediately post-HIIE (P0H), 3 hours post-HIIE (P3H), and 24 hours post-HIIE (P24H). Transcriptomic analysis was employed to identify broad patterns in gene expression related to autophagy/mitophagy in skeletal muscle. The largest number of differentially expressed genes was observed in hypoxia after exercise 24 hours. PCA analysis did not show any significant difference between exercise conditions. Autophagy-animal and mitophagy-animal pathways (not ranked in the top 20) were significantly enriched only in hypoxia 24 hours post-exercise, and the key signaling pathway (mTOR signaling pathway) did not exhibit pronounced changes at all conditions after HIIE. To focus on autophagy/mitophagy-related genes/pathways, I chose 604 genes related to autophagy/mitophagy as target genes to perform differential expression analysis. Again, the largest number of differentially expressed genes related to autophagy/mitophagy was observed in hypoxia after exercise 24 hours. However, recognized autophagy markers such as LC3II, LC3II/LC3I ratio, P62, and mitophagy receptors (NIX, BNIP3) did not show significant differences in expression level in all conditions, and mitophagy receptor FUNDC1 was observed in hypoxia, indicating a lack of robust change in autophagy/mitophagy after a single session of HIIE with and without hypoxia. Similar expression patterns were only observed between different time points under the same exercise condition. Although our result suggests an elevated level of genes related to autophagy/mitophagy pathways under hypoxia exercise, it did not correlate with physiological performance, as a higher percentage of HRpeak and V̇ O2peak, and a higher RPE were observed in both NR and HY compared to NA. Furthermore, the increased gene expression level in hypoxia is likely affected by time points after exercise. Our result suggests that activation of autophagy/mitophagy pathways is unlikely to be responsible for the difference in observed physiological response.

Unaccustomed, strenuous exercise can cause skeletal muscle damage that subsequently induces an acute inflammatory response in the tissue which is marked by an infiltration of leukocytes into the damaged muscle. To try and suppress the initial pro-inflammatory response in skeletal muscle of horses performing a single exercise stress test, a commercial sodium hyaluronate (HA) treatment was administered and tested for anti-inflammatory properties. Unfit, adult Thoroughbreds were intravenously injected three times with HA or received no injection at all (CON) over a 3-week period before performing a single submaximal exercise test. Gluteal muscle biopsies were collected before and 1 h after the completion of exercise for RNA-Seq and staining. The results indicated that HA treatment in horses down regulated genes associated with lymphocyte activation and cytokine production (Il17RA, OSCAR, LYL1, TLR1, TLR2, TLR8, TLR10) but did not irreversibly down regulate these genes with the addition of exercise. Exercise as a stressor did cause an acute inflammatory response in muscle which was seen through global expression of macrophage and neutrophil surface markers (NCF2, ELANE, CD168I). These results determine that HA treatment does act as an anti-inflammatory in equine skeletal muscle but does not possess prolonged effects with the initiation of inflammation.

Ribonucleic acid (RNA) molecules are traditionally known as an intermediate product in gene expression, but they are a diverse group of biomolecules with wide-ranging roles in regulating cell state. High-throughput RNA-sequencing technologies have enabled sensitive measurements of the collection of RNA molecules in cells, also known as the transcriptome. More recently, single-cell transcriptomics methods have been adapted to record the spatial location of each RNA molecule. Spatial transcriptomics gives context to measurements of gene expression and reveals patterns of gene regulation. Single-cell and spatial transcriptomics represent a powerful tool kit to study any biological system. The modularity of transcriptomics measurements enables integration of data from many disjointed experiments. We leveraged this facet of single-cell transcriptomics to jointly analyze single-cell transcriptomics data from more than 100 individual samples, together representing more than 365,000 single-cell transcriptomes. We used this data set to study rare cell types in skeletal muscle regeneration. We then applied low-resolution spatial transcriptomics to understand the spatiotemporal patterns of gene expression in skeletal muscle regeneration and used our harmonized transcriptomic reference to achieve a local deconvolution of cell subtypes. This analysis identified the spatiotemporal variation in cell subtype colocalization during injury recovery. Next, we extended these data with a new molecular strategy to broaden the repertoire of RNAs which can be captured by existing spatial transcriptomics platforms. Spatial total RNA-sequencing (STRS) leverages in situ polyadenylation of non-A-tailed RNAs to capture noncoding and non-host transcripts which are missed by standard methods. We applied STRS to a time course of muscle injury response and identified patterns of noncoding RNAs which localize to the injury. We then used STRS to spatially co-map viral RNAs and the host gene expression response in a mouse model of viral myocarditis. Finally, we review the state of transcriptomic technologies in skeletal muscle research and discuss the potential impacts of emerging technologies. Here we also include an updated resource with more than 2.5 million single-cell and single-nucleus transcriptomes from either mouse or human musculoskeletal tissues. Together, these efforts demonstrate new molecular and computational strategies to extend transcriptomics technologies to new cell types, new analytes, and new applications.

Glycogen synthesis in skeletal muscle (SM) is responsible for the majority of post-prandial glucose disposal in healthy individuals. In type 2 diabetes mellitus (T2DM), compromised skeletal muscle glycogen synthesis contributes to hyperglycemia. L6 rat SM cells can develop insulin resistance with prolonged insulin stimulation and provide an in vitro model for mechanistic dysfunction in glycogen homeostasis. SM glycogen is synthesized by glycogen synthase (GS) in response to insulin stimulation. Canonically, insulin signaling increases GS activity through inhibition of its Ser641 regulator glycogen synthase kinase-3β (GSK3β) by protein kinase B (AKT). Proximal insulin signaling activates AKT via Ser473 phosphorylation which proceeds to inhibit GSK3β via Ser9 phosphorylation. All-trans-retinoic acid (RA) has been reported to enhance insulin stimulated glycogen accumulation after 6 days of treatment in terminally differentiated L6 myotubules. We hypothesized that RA would also enhance insulin stimulated glycogen accumulation during myoblast differentiation through altered AKT-GSK3β-GS regulation. Differentiating L6 myoblasts treated with RA (0.5 µM) and insulin (10 nM) were harvested after 2 and 6 days of treatment. We reported that RA significantly enhanced insulin stimulated glycogen accumulation after 2 days of treatment without affecting pAKT (Ser473), pGS (Ser641), or pGSK3β (Ser9) expression. After 6 days of treatment, insulin induced glycogen accumulation as well as the expression of both pGS (Ser641) and pGSK3β (Ser9) without additional effects from RA. We proceeded to investigate the alterations of these key glycogen regulators further using the protein synthesis inhibitor cycloheximide (CHX) during a dose response time course study. CHX dramatically induced the expression of pGSK3β vi (Ser9) and pGS (Ser641) between 24 and 48 hours, indicating a shift in GS regulation away from GSK3β. CHX (10 µM) also inhibited GSK3β expression, dramatically induced pGSK3β (Ser9) expression, and attenuated RA (0.5 µM) and insulin (10 nM) stimulated glycogen accumulation after 48 hours. In contrast, CHX (10 nM) induced glycogen accumulation after 3 hours of treatment in rat SM ex vivo but did not alter expression of key regulatory proteins. The main findings of this dissertation indicate that the currently understood mechanisms of glycogen homeostasis in SM are far from complete.

The genetic selection for high growth rate, breast muscle yield, and feed efficiency in modern broilers has been a double-edged sword. While it has resulted in the desired traits, it has also led to broiler myopathies. Recent omics studies have shown that hypoxia may be a potential factor that damages the myofiber niche and compromises muscle repair processes. The objective of this study was to optimize isolation protocols for primary broiler satellite cells (SCs) and fibroadipogenic progenitors (FAPs) and to characterize SCs proliferation and differentiation under hypoxia. The study first focused on the optimization of isolation procedures and culture conditions for primary broiler SCs. Simple and cost-effective preplating techniques were used to purify SCs, which may improve the fidelity of follow-on experiments. In addition, myogenic differentiation potential was maximized under optimal conditions with different types and concentrations of serum and chicken embryo extract. This dissertation further demonstrated the effects of hypoxia on the differentiation and metabolism of SCs. Satellite cells were exposed to hypoxia conditions for 48 h and normoxia recovery. Hypoxia improved SCs proliferation by maintaining Pax7 and reducing Myod1 and myogenin while reducing myogenic differentiation. The cell proliferation was not rescued by 24 h normoxia recovery, whereas myogenic potential was recovered. In addition, hypoxia-activated AMPK and upregulated proteins involved in lipid metabolism and cellular stress, with decreased mitochondrial activity and increased lipid accumulation in myotubes, which indicates that the balance between fatty acid uptake and oxidation was disrupted. Several differentially expressed genes were also identified across myogenic differentiation under hypoxia with targeted RNA sequencing. Lastly, we established an isolation protocol for broiler FAPs for the first time. Through the comparative analysis between FAPs and preadipocytes, the induction of adipogenic differentiation and morphology of adipocytes derived from muscle and adipose tissues are similar. However, fatty acid methyl ester and targeted RNA sequencing data revealed a distinct separation between FAPs and preadipocytes throughout adipogenic differentiation. In conclusion, these data provide valuable insight into the biology of SCs and FAPs and the effects of hypoxia on SC proliferation and differentiation to better understand broiler myopathies.

Although skeletal muscle displays an astonishing regenerative capacity, injuries or diseases that resulted in bedridden or chronic muscle wasting can overwhelm this intrinsic feature of skeletal muscle and lead to functional deficit (range of motion and/or strength) and overall reduction in quality of life. Microenvironmental cues within injured skeletal muscle dictate regenerative and repair process which are tightly coordinated interplay among resident cells, cells recruitment and immune response following an assault in the muscle extracellular matrix (ECM). The successful regeneration of functional tissues requires both appropriate modulation of the inflammatory response, and activation of a variety of cell populations. Biomaterials offer unique opportunities to spatiotemporally control cytokine delivery and may provide significant benefits in the modulation of immune cells and muscle-immune micro-environment (MIME) to promote regeneration. The aim of this dissertation is to create injectable therapeutics that are capable of both modulating the inflammatory response and directly promoting muscle regeneration. In aim 1, we investigated the safety and therapeutic potential of human muscle derived decellularized ECM to promote muscle regeneration in mouse disuse and reloading hindlimb injury model and rabbit chronic rotator cuff tear muscle degenerative injury model. In aim 2, we investigated the therapeutic potential of delayed delivery of IL-10 to boost muscle regeneration when combined with progenitor cells repair (mined muscle grafts) in rat volumetric muscle loss injury model. Together, the findings of these two aims further our understanding of the role that pro-regenerative immune cells play in promoting endogenous mechanisms of tissue repair after muscle injury and present potential therapeutic avenues to modulate the MIME in aiding muscle repair and recovery.

Huntington’s disease (HD) is a genetic disorder associated with progressive cognitive and motor decline. Recent studies in HD models suggest primary peripheral pathologies arise independent of changes in the central nervous system. Our lab found defects in skeletal muscle occurring early in the progression of disease in transgenic R6/2 HD mice, resulting in membrane hyperexcitability. Additionally, there is evidence of decreased quantal content in late-stage R6/2 mice. Here, we investigate pre-synaptic and post-synaptic function at single neuromuscular junctions (NMJ) to make direct comparisons of disease progression in the muscle membrane and motor nerve terminal. We hypothesize that muscle membrane defects will precede reductions in neuromuscular quantal content during the progression HD in R6/2 mice. Muscle membrane properties and synaptic transmission were measured in 8-, 10-, and 12-week R6/2 mice. Identifying the earliest pathological influence of dysfunction at the NMJ may provide targets for treatment of motor symptoms in HD.

Excitation of skeletal muscle cells triggers a large voltage spike known as an action potential (AP), leading to muscle contraction. Modeling of an AP is typically done using the method developed by scientists Hodgkin and Huxley (HH). In the HH method, voltage and time gated Na+ and K+ ionic currents are simulated, along with a positive “Leak” ionic current and capacitive current. Due to the complexity and the computational time required for simulation, direct fitting of HH parameters to experimental APs has rarely been attempted. A previous thesis at Wright State performed direct fitting for the case of a single compartment muscle cell. This study will introduce propagation to the existing model by adding small longitudinal segments simulating the currents flowing and triggering APs in later portions of the cell. If no adjustments to the single compartment HH parameters are made, adding increasing longitudinal segments leads to a very poor simulation of the AP shape. The simulated AP is too wide, and the peak voltage is too low. However, very few clear links between the number of segments and changes in specific HH parameter values were identified. Each parameter was found to vary by at least a factor of 2 between similar data sets. It is clear that multiple parameter sets are allowable, obscuring direct links between segments and parameter values. In order to further understand the robustness of the found parameter sets and to identify what regions of parameter space is allowable in order to achieve a well fit AP a confidence interval test was completed. Scanning through each parameter and fitting the rest revealed that each of α_h_bar, β_h_bar, kβh, V_n_bar, kαn, α_n _bar, β_n_bar, and Na permeability hold a well-defined interval in which near perfect fits can be found. While for kαh, kβn, and kβm no upper-bound was identified, and for K permeability, V_h_bar, kαm, β_m_bar, α_m_bar, V_m_bar the patterns where unclear.

Skeletal muscle fibrosis is a common characteristic of different pathologies, including aging, muscular dystrophy, neurodegenerative disease, and severe extremity trauma. The fibrotic supplantation of muscle tissue impacts quality of life through disabilities ranging from reduced function and loss of mobility to osteoarthritis and elected limb amputation. It is the result of a dysregulated immune response that drives excessive extracellular matrix deposition and impairs the tissue resident muscle stem cells (MuSCs), leading to aberrant or muted muscle regeneration, degeneration of neuromuscular synapses, and muscular atrophy. However, the transcriptional networks and epigenetic changes that diminish MuSC function, how MuSC impairments impact innervation, and how exacerbated inflammatory and fibrotic signaling hinder skeletal muscle repair remain poorly understood. This dissertation seeks to elucidate the cellular and molecular mechanisms governing MuSC dysfunction and skeletal muscle fibrosis using high throughput, high dimensional methodologies in combination with various animal models of disease and injury to spatiotemporally profile MuSCs and their microenvironment in regenerative and fibrotic contexts. The first two studies employ a MuSC-centric approach to understand intracellular signaling dysregulation in age and neuromuscular disease and how it functionally impairs MuSC ability to maintain skeletal muscle homeostasis. First, integration of transcriptomics and chromatin accessibility datasets generated from aged and young murine MuSCs across a regenerative injury time course revealed that aging elicits multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility, and patterns of transcription factor binding activities. Both in vivo treatment of MuSCs with retinoic acid and silencing the expression of the myogenic co-factor, DNA damage inducible transcript 3, helped restore aged MuSC state and function towards that of young MuSCs. We then showed that following nerve injury, a subset of MuSCs engrafts proximally to the neuromuscular junction (NMJ) in healthy young adult muscles, but that with age or neuromuscular degeneration induced by CuZn superoxide dismutase knockout (SOD1-/-) this localized engraftment behavior is reduced, suggesting that MuSCs lose their ability to support synaptic integrity in these contexts. Single cell transcriptomics of murine MuSCs revealed a subset in aged animals and SOD1-/- animals that shares similarity with synaptic myonuclei, which may reflect a reduced fusion capacity and thus reduced ability to contribute to NMJ regeneration. Moreover, genetically rescuing the motor neurons via over-expressing human SOD1 only in the motor neurons reverted the MuSC response towards that observed in young animals. The final three studies enhance our understanding of the cellular and molecular mechanisms driving the fibrotic degeneration that occurs following severe injury. Using spatial and single cell transcriptomics as well as metabolipidomics and immunofluorescent techniques, we revealed exacerbated and persistent inflammatory and fibrotic signaling mediated by neutrophils, scar-associated macrophages, and fibro-adipogenic progenitors, which is partially antagonized by cytolytic natural killer cells. Elevated transforming growth factor beta (TGF inhibition reduced neutrophil accumulation, partially rescued functional recovery, and reduced collagen accumulation. Finally, restoring the balance of pro-resolving to pro-inflammatory lipid mediators by repleting Maresin 1, a pro-resolving lipid mediator, reduced macrophage and neutrophil accumulation, boosted MuSC proliferation, reduced TGF 1, and improved regeneration. Collectively, this work facilitates a deeper understanding of the regulation that tissue resident stem cells utilize during aging and healing, reveals unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors, and elucidates microenvironmental factors that impede regeneration following traumatic injury.