Excessive alcohol consumption is a leading cause of death and disability globally and can lead to diseases such as alcoholic skeletal myopathy. Skeletal muscle is the largest organ in the human body and functions to regulate whole-body energy homeostasis. Additionally, skeletal muscle can function as an endocrine organ via secretion of myokines. Two myokines, myonectin and irisin, present a wide range of effects upon metabolism, inflammation, and tissue survival- signaling. We hypothesized that chronic alcohol consumption will result in reduced circulating myonectin and irisin levels. Mice were fed an ethanol-containing or control diet for 10 days or 6 weeks. Tissues and serum were collected from mice and immunoblotting was used to quantify myonectin and irisin levels. Our data demonstrated that neither a 10 day nor 6-week ethanol diet was effective in altering myonectin levels, whereas irisin was undetectable. Therefore, we conclude that these myokines are not affected by alcohol consumption.

The Liver Kinase B1 (LKB1)/AMP-Activated Protein Kinase (AMPK) signaling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm. Two methods of AMPK activation were used to characterize LKB1/AMPK signaling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control (C) mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation (ES) of the diaphragm. Diaphragms were excised 60 minutes post-AICAR injection and immediately after ES. AMPK phosphorylation increased with AICAR and ES in C but not KO mice. Acetyl CoA carboxylase (ACC) phosphorylation increased with AICAR in C but not KO mice, but increased in both genotypes with ES. While the majority of mitochondrial enzyme levels were lower in KO diaphragms, uncoupling protein 3 (UCP-3) levels were not different between genotypes. A IIx to IIb fiber type switch was observed in KO diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover. In conclusion, LKB1 regulates AMPK phosphorylation, mitochondrial enzyme expression, fiber type distribution, as well as recovery of the diaphragm from fatigue.

Skeletal muscle development and function is governed by a conserved set of Transcription Factors (TFs) that regulate gene expression. The TF gene regulation is stimulus driven and cell-type and time point specific. TBX5 is an essential dosage sensitive regulator of heart and limb development. In the skeletal system, TBX5 is expressed in early stages in the lateral plate mesoderm and gives rise to the forelimb. TBX5 is also involved in proliferation and differentiation and survival pathways in both heart and limb development. Mutations in TBX5 gene lead to HOS which is characterized by various types of cardiac and musculoskeletal defects. TBX5 mechanism of action and its spatiotemporal function in skeletal muscle development has yet to be fully understood. TBX5 regulation is controlled through various factors such as alternative splicing, protein-protein interactions, Post-Translational Modifications (PTMs) and microRNAs. To date, many TBX5 interactors have been identified in cardiac cells however TBX5 protein interactors and target genes in skeletal muscle cells have not been studied. Understanding the protein interactome of TBX5 in skeletal muscle will enhance the current understanding of its mechanism of action. In this study we have characterized TBX5 with focus on its regulation, expression and biochemical properties in cardiac and skeletal muscle cells and moreover its mechanism of action specifically in skeletal muscle proliferation and differentiation. Chapter 1 discusses TBX5 regulation through alternative splicing leading to the existence of 5 distinct TBX5 isoforms with variable transcriptional activity, cardiac and limb expression pattern, biochemical properties and function. We show the pro-proliferation role of TBX5a in myoblasts while TBX5c shows to be pro-differentiation leading to the formation of myotubes in skeletal muscle C2C12 myoblasts. This opposing role of the two TBX5 isoforms lead us to studying TBX5 mechanism of action in proliferation and differentiation of skeletal muscle cells. In this study using a mass spectrometry-based approach we have identified novel TBX5 interacting partners in skeletal muscle cells for the first time by using stably overexpressed 3xFlag TBX5 via retroviral transduction in C2C12 cell line. Nuclear protein extracts were immunoprecipitated and sent for HPLC-ESI-MS/MS to identify potential protein partners of TBX5 in skeletal muscle cells. Moreover, the same stable cell line was used to identify TBX5 downstream target genes in these cell types by sending RNA extracts for microarray analysis. Amongst the 200 protein interactors identified, MYBBP1a and TBX5 interaction was confirmed and studied. The microarray analysis identified over 1200 differentially expressed genes and potential downstream targets of TBX5a from which Myostatin (Mstn) and Cyclin D2 (CcnD2) were both significantly upregulated and further confirmed and studied in relation to proliferation and differentiation in skeletal muscle cells. Chapter 2 focuses on the cooperative interaction between TBX5a and MYBBP1a inhibiting muscle specific gene promoter, Myogenin (MyoG). TBX5a and TBX5c seem to both interact with MYBBP1a but result in variable transcriptional activity of both MyoG and Mstn gene promoters. We show that TBX5 is upstream of Mstn, it binds to the promoter on specific TBE sites, and is able to upregulate Mstn promoter activation. In vivo, we show that MDX mice limb skeletal muscle tissues show elevated levels of TBX5, MYBBP1a and MSTN expression which suggest that the TBX5 pathway is associated with and indicative of the onset of proliferation and regeneration in MDX skeletal muscle tissue. Chapter 3 discusses the role of TBX5 in proliferation and regeneration of skeletal muscle cells by identifying that TBX5 binds to CcnD2 promoter and upregulates its activation which is a known cell cycle gene critical in cell proliferation and survival. Moreover, we identify GATA4 as a TBX5a cofactor in myoblast proliferation and show synergistic activation of Ccnd2 promoter by cooperative TBX5a and GATA4 action. We further show that Tbx5 heterozygote mice exhibit decreased levels of CCND2 and other proliferation markers, as well as decreased expression of PAX7 (marker of satellite cells) compared to WT skeletal muscle tissues. We also show that the heterozygous loss of Tbx5 impairs the process of regeneration in a cardiotoxin-induced injury model in mouse limb tissues. Tbx5 heterozygote mice exhibit less proliferation and impaired regeneration 4 days after injury, followed by decreased formation of regenerated fibers by 7 days post-injury compared to the wildtype mice skeletal muscle tissues; suggesting that TBX5 function is important in maintaining adult muscle regenerative capacity. Together, this study has characterized TBX5 isoforms and identified novel TBX5 protein partners and targets in the skeletal muscle cells and sheds light on TBX5 regulatory mechanism in proliferation and differentiation of skeletal muscle cells and its potential implications in HOS and other muscular diseases.

No description available.

Over the past 20 years, our collaborative research team at the Ottawa Hospital and the University of Ottawa have extensively investigated molecular and metabolic differences between individuals with obesity in the highest (DS) and lowest (DR) quintiles for rate of weight loss following 6 weeks of caloric restriction. Research on these cohorts of individuals with extreme phenotypes in diet-response has revealed that DS individuals have several skeletal muscle metabolic advantages, including increased proportions of type I oxidative fibres, increased mitochondrial proton leak, enhanced fatty acid metabolism, and a greater antioxidant capacity. Regular physical exercise provides a vast array of beneficial effects to metabolic health, including increases in skeletal muscle mitochondrial bioenergetic capacity and muscle cross-sectional area, leading to the hypothesis that exercise may be particularly beneficial to women with diet-resistant obesity. The overall aim of this thesis was to determine whether six weeks of exercise training improves skeletal muscle mitochondrial function and attenuates chronic low-grade inflammation in women with obesity previously identified as diet-sensitive (DS) and diet-resistant (DR). Here, we demonstrate that exercise training improves body composition, enhances cellular maximal respiration, and increases mitochondrial length preferentially in DR women. Contrary to our hypothesis, exercise training increased skeletal muscle IKK-NFκB inflammatory signaling to a greater extent in DR individuals, despite improvements in systemic cytokine concentrations. In response to an inflammatory challenge, LPS-treated primary myotubes derived from DR and DS skeletal muscle responded similarly and respiratory capacity was preserved. Taken together, these findings suggest that exercise can be especially beneficial as part of a treatment plan for DR individuals, and that DS and DR skeletal muscle have systemic and mechanistic differences in inflammatory responses.

The overall focus of this project has two parts: First, was to develop a protocol utilizing serum-free media formulations and defined plating and culture techniques to create functional in vitro myotubes derived from adult skeletal muscle satellite cells. The second was to manipulate the inherent muscle parameters such as force output and fatigue of these myotubes by employing exercise regimes or by small molecule application. The importance of serum-free medium use for in vitro cultures is becoming increasingly important in creating functional systems that can be validated for drug testing by the Food and Drug Administration (FDA). Also, the study of age related diseases as well as the potential for “personalized medicine” relies on the proliferation and maturation of satellite cells from adult derived tissue. For that purpose, a serum-free medium and culture system was designed to create mature striated myotubes in culture on a defined non-biological substrate N-1[3-trimethoxysilyl propyl] diethylenetriamine (DETA). These myotubes were evaluated by morphology, muscle specific protein expression, and by muscle functionality. After the thorough characterization of the resultant myotubes the functional output of the muscle was altered utilizing chemical means (creatine supplementation and PGC-1? agonists), chronic long term stimulation, and the use of PGC-1? deficient tissue. In this thesis presentation the utility of the newly developed medium formulation to create myotubes from a variety of adult derived muscle sources will be shown. A protocol in which to exercise skeletal muscle in vitro to alter endurance was developed and employed to manipulate skeletal muscle. Finally, small molecules were tested to validate this system for drug study use. This engineered system has the potential for high-throughput screening of drugs for efficacy and drug toxicity studies as well as general biological studies on muscle fatigue.

The purpose of this dissertation was to investigate the molecular events associated with the onset of skeletal muscle disuse in humans. Study I examined global gene expression changes in vastus lateralis muscle following 48h unloading (UL) and 24h reloading (RL) in humans. Results showed that functions related to protein degradation and oxidative stress were enriched following UL and that these global gene expression patterns were not readily reversed following RL, thus indicating that molecular events associated with short-term disuse may persist beyond the duration of the stimulus. In contrast to previous work in IM, collagen gene expression increased in this study, demonstrating that differences in molecular signaling may exist among disuse models in humans. Study II of this dissertation expanded on the findings of Study I to investigate global gene expression patterns related to the early stage of multiple disuse models. Microarray data collected 48h post-UL in Study I were analyzed within the context of data previously collected in our laboratory following 48h immobilization (IM) and spinal cord injury (SCI). Results showed that the disuse models shared a small subset of commonly differentiated genes. Furthermore, the similarities between IM, SCI, and UL extended beyond specific genes to include commonly enriched functions and pathways such as protein degradation and oxidative stress, suggesting that these molecular mechanisms are involved in the early stages of disuse, regardless of specific stimulus. In Study III, an in vitro model of skeletal muscle was used to test the exploratory hypothesis that induction of oxidative stress response gene heme oxygenase-1 (HMOX1) would lead to decreases in gene expression associated with proteolysis, namely ubiquitin E3 ligases atrogin1 and MuRF1, as well as increased XXT cleavage (a marker of metabolic enzyme activity). In this study, C2C12 myotubes were pre-treated with hemin (an inducer of HMOX1) and then treated with H2O2 to elicit oxidative stress. Results showed that hemin treatment resulted in increased HMOX1 expression and decreased E3 ligase expression. Furthermore, hemin-treated cells exhibited increased XTT cleavage compared to controls. HMOX1 may be a promising gene target to protect against oxidative stress that accompanies early stages of disuse.

PRMT biology during skeletal muscle disuse. / Protein arginine methyltransferase 1 (PRMT1), PRMT4 (also known as co-activator-associated arginine methyltransferase 1; CARM1), and PRMT5 are critical components of a diverse set of intracellular functions. Despite the limited number of studies in skeletal muscle, evidence strongly suggests that these enzymes are important players in the regulation of phenotypic plasticity. However, their role in disuse-induced muscle remodelling is unknown. Thus, we sought to determine whether denervation-induced muscle disuse alters PRMT expression and activity in skeletal muscle within the context of early signaling events that precede muscle atrophy. Mice were subjected to 6, 12, 24, 72, or 168 hours of unilateral hindlimb denervation. The contralateral limb served as an internal control. Muscle mass decreased by ~30% following 168 hours of disuse. Prior to atrophy, the expression of muscle RING finger 1 and muscle atrophy F-box were significantly elevated. The expression and activities of PRMT1, CARM1, and PRMT5 displayed differential responses to muscle disuse. Peroxisome proliferator-activated receptor-γ coactivator-1α, AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinase expression and activation were altered as early as 6 hours after denervation, suggesting that adaptations in these molecules are among the earliest signals that precede atrophy. AMPK activation also predicted changes in PRMT expression and function following disuse. Our study indicates that PRMTs are important for the mechanisms that precede, and initiate muscle remodelling in response to neurogenic disuse. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that is capable of adapting to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important players in the regulation of skeletal muscle remodelling. However, their role in disuse-induced muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the role of PRMTs within the context of early, upstream signaling pathways that mediate disuse-evoked muscle remodelling. We found differential responses of the PRMTs to muscle denervation, suggesting a unique sensitivity to, or regulation by, potential upstream signaling pathways. AMP-activated protein kinase (AMPK) was among the molecules that experienced a rapid change in activity following disuse. These alterations in AMPK predicted many of the modifications in PRMT biology during inactivity, suggesting that PRMTs factor into the molecular mechanisms that precede neurogenic muscle atrophy. This study expands our understanding of the role of PRMTs in regulating skeletal muscle plasticity.

Introduction Weight loss practices are commonly used by athletes who perceive lower levels of body fat and higher fat-free mass (FFM) as advantageous for physical performance. High protein diets have been associated with greater fat loss and greater retention of lean mass during periods of energy restriction (ER) in individuals with obesity. However, less is known about the effects of high protein diets in trained individuals during ER. It has been proposed that leaner individuals are more at risk of losing FFM during ER than individuals with overweight. The current ACSM recommendation for protein intake in active individuals is 1.2-1.7g/kg. However, it is unclear whether higher intakes are necessary to maximize FFM retention and maximize physical performance in athletes undergoing periods of ER. Objective Our primary objective was to determine whether high protein intakes could maximize FFM retention in trained individuals during periods of ER. We also aimed to determine whether higher protein intakes are superior at maintaining physical performance. Lastly, we aimed to determine whether high protein intakes could mitigate adaptive thermogenesis (AT) in response to ER. Methods Twelve college aged athletes (6 women and 6 men) from various sports were analyzed in this study. They underwent a 6-week 25% reduction in energy intake along side a 3-day full body resistance training program. Participants were randomly assigned to a low (~1.2g/kg), moderate (~1.6g/kg) or high (~2.2g/kg) protein group. Baseline and post-intervention measures were obtained via Dual X-ray absorptiometry (DXA), isotopic water dilution, indirect calorimetry, dietary records and strength based physical testing. Results Our results indicate a main effect of the intervention on fat mass reductions (19.66 ± 9.05 kg vs 18.02 ± 8.07 kg) (p = 0.016) despite non-significant reductions in body weight (p = 0.059). No significant changes in FFM were observed (p = 0.307). Significant increases in chest press 5RM strength (43.18 ± 14.95 kg vs 48.86 ± 17.46 kg) and chin-up maximal repetition (4.75 ± 5.64 vs 6.08 ± 6.56) were observed across all groups from baseline to post-intervention (p < 0.05). No significant increases in strength were noted on lower body movements. Resting energy expenditure (REE) was significantly reduced after the 6-week intervention (1743.52 ± 295.74 kcal vs 1655.18 ± 263.23 kcal) (p = 0.006). What is more, post-intervention measured and predicted REE were also significantly different (1655.18 ± 263.23 kcal vs 1747.92 ± 263.20) (p = 0.012). No significant effects of protein were noted on any of the outcomes. Conclusion Our preliminary results indicate that the ACSM recommendation of 1.2-1.7g/kg is sufficient for most athletes even during periods of ER to maintain FFM and physical performance. We also found the possibility of early AT in that population independently of protein intake. In fact, REE was reduced by ~100 kcal/day more than predicted despite minimal weight loss and relative preservation of FFM.

The objective of this thesis was to examine muscle function and myofiber physiology in skeletal muscles in those with type 1 diabetes (T1D) by investigating the effects of diabetic myopathy on these metrics of muscle health under various conditions: at rest, after exercise and with increasing age. These works recruited adults from surrounding communities with T1D and non-diabetic counterparts (i.e. controls) matched for age, sex, body mass index, and self-reported physical activity levels. We hypothesized that adults with T1D would exhibit decreased muscle function (i.e. lower maximal strength) and altered myofiber physiology in each of these conditions. At rest, we observed that those with T1D exhibited more fast-twitch fibers and fewer satellite cells. After exercise, T1D muscles recovered less strength, showed higher amounts of myofiber damage, and delayed satellite cell proliferation. With increasing age, adults with T1D exhibited exaggerated signs of muscular aging compared to age-matched controls in the form of more abundant hybrid fibers and type 1 fiber grouping. Finally, individuals with T1D exhibited higher baseline expression of myostatin, a negative muscle growth regulator, compared to controls. Overall, our work provides the first evidence in muscle dysfunction from humans with T1D at various ages and after damaging exercise. Our findings provide novel insights on muscle health and its contribution to overall health during this lifelong, debilitating disease. Our work aims to guide future clinical & exercise guidelines with the ultimate purpose of improving the lives of millions of individuals living with T1D. / Thesis / Doctor of Philosophy (PhD) / Type 1 diabetes (T1D) is a disease that affects millions of adults worldwide by harming their ability to manage blood sugar levels. Insulin therapy has allowed for longer and healthier lives but requires constant attention manage blood sugar levels. Over time, the quality of life declines because of complications from T1D. Muscle is able to control blood sugar levels through exercise, but little is known about muscle in those with T1D. Therefore, the purpose of this work was to examine skeletal muscle health in people with T1D who do not have other complications. We found that adults with T1D exhibit signs of aging in their muscles earlier than non-diabetic people. Also, we observed that muscle from young adults with T1D recovered slower from exercise. Finally, we learned that people with T1D have more of a muscle-shrinking protein. This is the first evidence of dysfunctional muscle fibers at rest, after exercise, and with age in adults with T1D. This work aims to improve future guidelines for millions of adults with T1D.