The objectives of this study were to determine: the effects of maternal melatonin (MEL) supplementation during late gestation on the histological and molecular regulation in the Longissimus dorsi (LM) muscle of fetal bovine offspring, composition and gene expression of fetal perirenal (PR) adipose tissue, and LM gene expression in postnatal offspring at birth and d 195 of age. Maternal supplementation of MEL during late gestation resulted in no difference in calf fetal body weight or birth weight. However, at d 195 of age, calves from MEL treated dams had an average body weight increase of 20 kg. Fetal LM weight and length tended to be increased in calves from MEL treated dams. Fetal gene expression of calves from MEL treated dams resulted in: increased LM adenosine monophosphate-activated protein kinase-α (AMPK) and decreased PR adiponectin (ADIPOQ), CCAAT enhancer binding protein alpha (CEBPA), proliferator activated receptor gamma (PPARg), and stearoyl-CoA desaturase (SCD). The improved metabolic status of LM coupled with the decrease in adipogenic gene expression, could result in calves from MEL treated dams having improved lean muscle accretion and reduced overall adiposity during postnatal development.

Skeletal muscle formation is among the most striking examples of cellular plasticity in animal tissue development, where mononucleated, lineage-restricted progenitor cells are epigenetically reprogrammed to produce multinucleated myofibers. While some mediators of epithelial-mesenchymal transition (EMT) have been shown to function in myogenesis, regulation of this process at the interface of multipotency and myogenic differentiation remains poorly understood. The long noncoding RNA (lncRNA) Meg3 is processed from the >200 kb Dlk1-Dio3 polycistron, and while many encoded miRNAs have been shown to regulate skeletal muscle differentiation, regeneration, and aging, the functional relevance of encoded lncRNAs in skeletal muscle remains elusive. Here, I demonstrate that Meg3 is enriched in proliferating post-natal myoblasts, where it epigenetically modulates aspects of cellular plasticity to facilitate myogenic differentiation in vitro, skeletal muscle regeneration in vivo, and safeguard myogenic identity. Chronic inhibition of Meg3 in C2C12 myoblasts compromised cytoarchitectural and transcriptomic cell-state transitions required for myogenic fusion and differentiation. These differentiation defects were primarily driven by TGFβ-dependent Snai2 activation, which correlated with irregular Ezh2 activity and abnormal epigenetic marks in differentiating C2C12 cells. Similarly, adenoviral Meg3 knockdown compromised muscle regeneration in vivo, which manifested as abnormal mesenchymal gene expression, fibrosis, and interstitial cell proliferation in the regenerating milieu. Comparison of Meg3-depleted C2C12 myoblasts and injured skeletal muscle to literature-derived gene sets suggest that Meg3-deficient samples deviate from controls towards abnormal transcriptional states, including immature satellite cell activation, muscle aging, and adoption of an osteoblast-like cell ontology. Thus, Meg3 regulates myoblast identity to maintain proper cell state transitions in postnatal myogenesis.

The objective of this study was to determine breed specific effects of nutritional insults during gestation on regulation of muscle and adipose tissue growth in beef cattle offspring during fetal and early postnatal development. Angus and Brahman heifers were randomly assigned to 1 of 2 dietary treatments consisting of 100% or 60% based on net energy requirements for gestating heifers. Nutritional treatments were imposed from day 50 to 180 of gestation. Fetuses harvested at 180 days of gestation had smaller frames and spared critical organs. Gene expression in several fetal tissues indicated potential for compensatory growth. Postnatally, Brahman offspring weighed less than Angus offspring and had smaller heads and heart girths along with decreased expression of growth and myogenic genes in the Longissimus dorsi. There were no differences in growth or myogenic gene expression within the Longissimus dorsi due to treatment. However, restricted animals had a decreased expression of PPARG.

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The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins are essential regulators of the classical and alternative NF-κB pathways. The NF-κB pathway has been shown to be an important regulator of myogenesis and plays a role in skeletal muscle disease, but the involvement of cIAP1 and cIAP2 has not been examined in healthy skeletal muscle. I sought to characterize skeletal muscle of the cIAP1-null and cIAP2-null mice. We show mice lacking cIAP1 exhibit decreased satellite cell numbers in the TA following cardiotoxin-induced injury and in the uninjured soleus muscle, suggesting cIAP1 may be important for satellite cell expansion. cIAP2 may play a role in fiber maintenance and homeostasis as we show cross- sectional are of cIAP2-null uninjured tibialis anterior fibers at 7 and 10 weeks of age were significantly smaller than wild-type fibers. Furthermore, cIAP1- and cIAP2-null mice subjected to in situ force experiments demonstrated altered twitch kinetics compared to wild-type controls in the soleus and EDL, suggesting fast and slow-twitch fibers are affected differently by loss of cIAP1 and cIAP2. Further work elucidating the downstream mechanisms by which cIAP1 and cIAP2 regulate skeletal muscle development and regeneration will be beneficial to the development of treatments for muscular disorders. In this regard, Smac mimetic compounds (SMCs) are small molecule inhibitors that target cIAP1/2 for degradation, thus provide a potential therapeutic treatment for muscular disorders.

Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, the biology of these enzymes during muscle development remains poorly understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C2C12 muscle cell maturation, assessed during the myoblast stage, and during days 1, 3, 5, and 7 of differentiation, was employed as an in vitro model of myogenesis. We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines and symmetric dimethylarginines, indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, this study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially-mediated mechanism, in driving the muscle differentiation program. / Thesis / Master of Science (MSc) / Protein arginine methyltransferases (PRMTs) are responsible for many important functions in skeletal muscle. However, significant knowledge gaps exist with respect to PRMT expression and activity during conditions of muscle remodeling. Therefore, the purpose of this Thesis was to investigate PRMT biology throughout skeletal muscle development. Mouse muscle cells were employed to examine characteristics of PRMT1, -4, and -5 at numerous timepoints during myogenesis. PRMTs exhibited distinct patterns of gene expression and activity during muscle maturation. A PRMT1 inhibitor (TC-E) was utilized to investigate the role of this enzyme during myogenesis. Muscle differentiation was impaired in TC-E-treated cells, which coincided with reduced mitochondrial biogenesis and respiratory function. Altogether, these results suggest a PRMT-specific pattern of expression and activity during myogenesis. Furthermore, PRMT1 plays a crucial role in skeletal muscle differentiation via a mitochondrially-mediated mechanism. Our study provides a more comprehensive view on the role of PRMTs in governing skeletal muscle plasticity.

Lysosomal storage diseases (LSDs) are metabolic diseases which occur as a result of a deficiency of one of the essential lysosomal enzymes, called glycohydrolases. A mutation in the gene encoding one of these enzymes leads to an accumulation of unwanted substrates, resulting in a variety of clinical manifestations. A common symptom found in LSDs is skeletal muscle dysfunction, which includes muscle weakness, atrophy and loss of muscle mass. The genes for lysosomal hydrolases are well characterized; however, much less is known about how mutations in these genes affect the cell and lead to the muscle dysfunction observed. One pathway of interest is autophagy; it has been shown to be essential for maintenance of skeletal muscles. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of Sialidosis, Sandhoff Disease, and GM1-Gangliosidosis in order to assess autophagy and its impact on myogenic differentiation in skeletal muscles. Our results demonstrated that autophagy is induced upstream (ULK1 phosphorylation) but is inhibited at the autophagosome to lysosome fusion (p62 upregulation) in LSDs. We also found that myoblast fusion and myogenic differentiation are impaired. We conclude that blocking autophagy impairs myogenic differentiation, which potentiates the muscle dysfunction observed in LSDs. This work highlights autophagy as a new pathway of interest and possible therapeutic target to alleviate muscle dysfunction in LSDs, and other similar neurodegenerative diseases. / Thesis / Master of Science (MSc) / Lysosomal storage diseases (LSDs) occur because of a deficiency of lysosomal glycohydrolases. A common symptom found in LSDs is skeletal muscle dysfunction. Little is known about how a deficiency of these enzymes leads to the clinical manifestations observed. However, one pathway of interest is autophagy. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of LSDs to assess autophagy and its impact on myogenic differentiation in skeletal muscles. We demonstrated that autophagy is induced and blocked, and that myoblast fusion and myogenic differentiation is impaired. We concluded that the induction and block of autophagy impairs myogenic differentiation, which potentiates muscle dysfunction.

Skeletal muscle can perceive cellular energy status and substrate availability and demonstrates remarkable plasticity in response to environmental changes. Nonetheless, how skeletal muscle and its resident stem cells (satellite cells; SCs) sense and respond to nutrient flux remains largely undefined. The dynamic post-translational modification O-GlcNAcylation has been shown to serve as a cellular nutrient sensor in a wide range of cells and tissues, yet its role in skeletal muscle and SCs remains unexplored. Here, we ablated skeletal muscle O-GlcNAc transferase (OGT), and thus O-GlcNAcylation, and found the knockout mice exhibited enhanced glucose uptake, insulin sensitivity, and resistance to high-fat diet induced obesity. Additionally, mKO mice had a 3-fold increase in circulating levels of interleukin-15 (IL-15), a potent anti-obesity cytokine, potentially through epigenetic regulation of Il15 by OGT. To further investigate if there was a causal relationship between OGT ablation and the lean phenotype, we generated muscle specific OGT and interleukin-15 receptor alpha (IL-15ra) double knockout mice (mDKO). As a result, mDKO mice had blunted IL-15 secretion and minimal protection against HFD-induced obesity. Together, these data indicate the skeletal muscle OGT-IL15 axis plays an essential role in the maintenance of skeletal muscle and whole-body metabolic homeostasis. As satellite cells (SCs) play an indispensable role in postnatal muscle growth and adult regenerative myogenesis, we investigated the role of O-GlcNAcylation in SC function. To this end, we conditionally ablated OGT in SCs (cKO) and found cKO mice had impaired SC proliferation, in vivo cycling properties, population stability, metabolic regulation, and adult regenerative myogenesis. Together these findings show that SCs require O-GlcNAcylation, presumably to gauge nutritional signals, for proper function and metabolic homeostasis. Another critical yet often neglected player in myogenesis are mitochondria. Traditionally depicted as a power plant in cells, mitochondria are critical for numerous nonconventional, energy-independent cellular process. To investigate the role of both mitochondrial energy production and alternative mitochondrial functions in myogenic regulation, we ablated ATP synthase subunit beta (ATP5b) and ubiquinol-cytochrome c reductase (UQCRFS1) in C2C12 myoblasts to disrupt mitochondrial ATP production and mitochondrial membrane potential, respectively. Ablation of UQCRFS1, but not ATP5b, impaired myoblast proliferation, although lack of either gene compromised myoblast fusion. Interestingly, addition of the potent myogenic stimulator IGF-1 rescued ATP5b fusion but could not override UQCRFS1 knockout effects on proliferation or differentiation. These data demonstrate mitochondrial ATP production is not the "metabolic switch" that governs myogenic progression but rather an alternative mitochondrial function. In summary, skeletal muscle and their resident stem cell population (SCs) both use O-GlcNAcylation, feasibly to sense and respond to nutritional cues, for the maintenance of metabolic homeostasis and normal physiology. A deeper understand of both muscle and SC metabolic regulation may provide therapeutic targets to improve global metabolism and muscle growth. / Doctor of Philosophy / Skeletal muscle is responsible for approximately 20% of basal energy expenditure and 70-90% of insulin-mediated glucose disposal, and as such changes in skeletal muscle metabolism and insulin sensitivity have profound impacts on whole body metabolism. Skeletal muscle is a plastic tissue that can perceive nutrient availability, which permits metabolic adaptations to environmental changes. Deletion of the nutrient sensing pathway O-GlcNAcylation in skeletal muscle (mKO) protected mice from high-fat diet induced obesity and ameliorates whole-body insulin sensitivity. Skeletal muscle can secrete myokines to elicit endocrine effects on other tissues in the body, and as such, we proposed perturbation of this nutrient sensing pathway in skeletal muscle alters myokine secretion to elicit responses in other metabolically active tissues to support its energy requirements. Indeed, circulating levels of interleukin-15, a potent anti-obesity myokine, increased 3-fold in mKO mice. To determine the contribution of IL-15 to the mKO phenotype, we used a genetic approach to blunt IL-15 secretion from skeletal muscle (mDKO), which partially negated the lean mKO phenotype. Our findings show the ability of skeletal muscle to "sense" changes in nutrients through O-GlcNAcylation is necessary for proper muscle and whole-body metabolism. Moreover, this nutrient sensing mechanism is also important for proper muscle stem cell function, also known as satellite cells (SCs). Loss of O-GlcNAcylation in SCs impairs their ability to regenerate muscle after injury, which can be attributed to a reduced capacity to proliferate and an inability to maintain a healthy SC population. Interestingly, SCs lacking O-GlcNAcylation have a greater mitochondrial content. Using a myoblast cell line, we investigated the contribution of mitochondria to myogenesis, the formation of muscle, and found mitochondrial energy production is dispensable in the myogenic process. Our studies show skeletal muscle and SCs rely on highly integrated signaling cascades that sense and respond to intrinsic metabolic changes and extrinsic nutritional cues to function properly.

Thoroughbred racehorses undergo strenuous exercise which often leads to the occurrence of exercise-induced pulmonary hemorrhage (EIPH), in which capillaries rupture within the alveoli in the lungs causing bleeding. Severe cases of EIPH lead to epistaxis and may result in fatality. Presently, the loop diuretic furosemide is the only medication approved to mitigate the effects of EIPH. Often regarded in the racing industry as "performance enhancing" due to 4% weight loss ensued by its diuretic effect, it is unknown what effects furosemide may have on muscle recovery. Therefore, the objective of this study was to determine the effects various doses of furosemide may have on equine satellite cell (eqSC) myogenesis and metabolism. Mitotic index was increased (P<0.05) as a result of treatment with 100 µg/mL furosemide, a 10-fold pharmacological dose, in comparison to vehicle, but was not different (P>0.05) compared to the physiological dose of 10 µg/mL furosemide. Average cell number decreased (P<0.05) in the excess furosemide group compared to all other groups. Pax7 expression did not differ (P>0.05) between groups. Expression of the differentiation transcription factor myogenin, and embryonic sarcomeric myosin heavy chain decreased (P<0.05) when cells were treated with 100 µg/mL furosemide. Fusion index and myotube area decreased (P<0.05) as a result of treatment with excess furosemide. Glycogen concentration in myotubes was lower (P<0.05) following treatment with 100 µg/mL furosemide, while IGF-1 was unsuccessful in rescuing the effects of furosemide. Excess furosemide decreased expression of muscle creatine kinase while increasing expression of phosphoglucomutase 1, glycogen synthase 1, and glycogen branching enzyme 1 (P<0.05). Excess furosemide decreased basal oxygen consumption rate (OCR) and increased OCR after addition of oligomycin (P<0.05). Excess furosemide did not affect myotube glycolysis rates in vitro. In conclusion, furosemide inhibits muscle differentiation and oxidative metabolism in eqSCs. / Master of Science / Thoroughbred racehorses often bleed from the lungs as a result of high-intensity exercise. This condition can oftentimes be fatal depending on severity. Furosemide, is used in the industry to reduce blood pressure within the lungs during racing to prevent bleeding. Furosemide, a diuretic given four hours prior to a race, causes a horse to excrete up to 4% of its body weight. This effect of furosemide decreases the weight a horse must carry during a race, thus allowing the horse to run faster. Therefore, deemed as a performance enhancing drug due to its effects on the kidney, to our knowledge, no research has been conducted on what effects furosemide might have on muscle generation. High-intensity exercise causes massive muscle damage and therefore must be repaired to prepare for the next bout of exercise. Muscle generation is called myogenesis. Stem cells, or satellite cells, that lie within the muscle become activated, recognizing the need for muscle repair. Satellite cells divide, increasing in cell number and then fuse together, forming new muscle fibers. Satellite cells undergo different types of metabolism depending on their state of development. For example, proliferating cells require glucose for energy, while cells fusing together forming myotubes, require oxidative metabolism for long-lasting energy. Therefore, the objective of this study was to determine the effects furosemide might have on muscle formation and metabolism. The excess furosemide dose (100 µg/mL) decreased cell proliferation. The expression of regulatory factors responsible for forming myotubes at different stages of muscle development are decreased when cells were treated with the defined excess furosemide dose. Furosemide decreased the ability of satellite cells to generate myotubes. Glycogen concentration was also decreased as a result of excess furosemide treatment. Gene expression of enzymes involved in glycogen synthesis were increased from treatment with our excess furosemide dose. No effect of furosemide was seen on glycolysis, whereas oxidative metabolism suffered as a result of treatment with excess furosemide. In conclusion, furosemide does indeed affect muscle generation and oxidative metabolism.