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This study investigated the effect of bone marrow derived mesenchymal stem cells (MSCs) in a PEGylated fibrin gel (PEG) seeded into a decellularized extracellular matrix (ECM) on recovery of skeletal muscle following a volumetric muscle loss (VML) injury. Six to nine month old male Sprague-Dawley rats were used in this study. Approximately one-third of the skeletal muscle mass of the lateral gastrocnemius (LGAS) was removed from the LGAS, which was immediately replaced with an acellular ECM of the same dimensions. Seven days after injury, animals were injected with one of four solutions: saline (SAL), MSCs (MSC), PEGylated fibrin hydrogel (PEG), or MSCs in PEG (PEG+MSC). Maximal isometric tetanic tension (Po) of the LGAS was assessed fifty-six days after VML injury, followed by histological evaluation. VML injury resulted in a functional impairment of the LGAS capable of producing 76.1± 4.9% of the force generated in the non-injured contralateral LGAS. Tetanic tension of the PEG+MSC treated group was significantly higher compared to all other treatment groups (p < 0.05), although specific tension (N/cm2) in the PEG+MSC group (79.7±4.0%) was only significantly higher compared to SAL (58.2±3.0) and PEG (64.0±2.1%) treated groups (p < 0.05). However, LGAS mass was significantly higher in the PEG+MSC group compared to all other groups (p < 0.05). These findings suggest the combination of the PEG+MSC did not lead to a significant increase in muscle function compared to MSC treatment alone, and demonstrates the importance of MSCs in skeletal muscle regeneration in VML injury models. However, as evident by the significant increase in LGAS mass, PEG+MSC treatment may lead to histological differences not evaluated in this study. Gross morphology of the repaired gastrocnemius was indistinguishable from the contralateral control. / text

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.

Skeletal muscle has a remarkable capacity for regeneration, mediated by muscle stem cells that can self-renew or differentiate to form the mature myofibers of the tissue. Several human diseases are characterized by a loss of function and strength in skeletal muscle, with impairments in the ability to regenerate and consequent decreases in quality of life and increases in mortality. The work in this dissertation has focused on developing methods for combating muscle disease. This goal has been approached through attempts at cell replacement therapy - by generating muscle cells that can be engrafted in vivo. I also investigated the influence on regeneration of the skeletal muscle microenvironment (skeletal muscle-resident fibroblasts), and systemic environment (inflammation in myogenic and non-myogenic tissues), both of which were found to affect skeletal muscle stem cell behavior and the efficiency of myogenic regeneration. Ultimately, these studies identified novel factors that impair or improve skeletal muscle differentiation, and that offer the potential to modulate the process of muscle regeneration. In the process of investigating if induced pluripotent stem cells from skeletal muscle retain an epigenetic memory conducive to myogenic differentiation, I discovered that precursor cells in skeletal muscle reprogram to a pluripotent state more efficiently. However, these induced pluripotent stem cells, like embryonic stem cells, retain strong barriers to skeletal muscle differentiation. Together, these findings offer insights into the process of muscle regeneration, and suggest new potential pathways towards treatment of muscle disease.

Skeletal muscle (SkM) possesses an impressive ability to regenerate in response to injury or chronic disease. This regenerative capacity is attributed to its resident mononuclear myogenic progenitors. Previous studies have identified several types of myogenic progenitors within SkM, some of which are isolated by fluorescence activated cell sorting (FACS) using cell surface markers. Studies in our laboratory have identified melanoma cell adhesion molecule (MCAM) as a cell surface marker expressed by myogenic progenitors in human fetal SkM. However, the relationship between MCAM expression and the degree of myogenic commitment of distinct MCAM+ populations has not been elucidated. In the present study, subpopulations of MCAM+ cells were purified by FACS on the basis of Hoechst 33342 dye uptake. Specifically, MCAM+ side population (SP) was isolated by Hoechst exclusion and MCAM+ main population (MP) on Hoechst incorporation. Sorted populations were first optimized for growth in vitro since SkM SP cells are difficult to maintain in culture. In particular, Invitrogen’s StemPro® MSC SFM medium was found to support propagation of human fetal SkM SP cells with minimal differentiation. Following this optimization, sorted populations were assessed for expression of myogenic markers before and after propagation and then for fusion potential in vitro and engraftment potential in vivo. The MCAM+ subpopulations were found to express myogenic markers to a significantly greater extent than MCAM- subpopulations. Furthermore, the MCAM+ subpopulations fused robustly into myotubes in vitro whereas the MCAM- subpopulations did not. Interestingly, the MCAM+ SP population exhibited the highest fusion potential in vitro and was the only MCAM+ subpopulation to engraft into dystrophic muscle in vivo following propagation. These results indicate that MCAM is associated with myogenicity and can be used to prospectively isolate a pure myogenic fraction from human fetal SkM tissue. Moreover, the MCAM+ SP retain its myogenic potential to a greater extent than MCAM+ MP after propagation. This suggests that the MCAM+ SP fraction contains a higher percentage of early myogenic progenitors compared to the MCAM+ MP fraction. Additional studies on MCAM-expressing populations in human fetal SkM may elucidate a potent population for use in cell-based therapeutic strategies for treating muscle diseases.

Lung tumors secrete multiple factors that contribute to cachexia, a severe wasting syndrome that includes loss of muscle mass, weakness, and fatigue. 80% of advanced lung cancer patients experience cachexia, which cannot be reversed by nutritional interventions, diminishes response to and tolerance of cancer treatments, and increases morbidity and mortality. Despite a multitude of clinical trials, there are currently no approved treatments. This deficiency suggests that not all of the factors that contribute to cachexia have been identified. Cancer is frequently accompanied by an increase in cyclooxygenase-2 (COX-2), a hallmark of inflammation. Clinical trials for COX-2 inhibitors have resulted in restoration of muscle mass and decreased fatigue. Along with loss of myofibrillar proteins, cachexia also induces mitochondrial dysfunction, which contributes to fatigue. The amelioration of fatigue by COX-2 inhibition suggests possible alterations to mitochondrial function. We hypothesized that there were unidentified tumor-derived factors that contribute to cachectic wasting and fatigue. Treatment of C2C12 myotubes with Lewis lung cancer-conditioned media (LCM) resulted in increased COX-2 content, myosin loss, and mitochondrial dysfunction. Mass spectrometry revealed 158 confirmed proteins in LCM. We focused on extracellular 14-3-3 proteins because they bind and regulate over 200 known partners. We found that depletion of extracellular 14-3-3 proteins diminished myosin content. CD13, an aminopeptidase, is the proposed receptor for 14-3-3 proteins. Inhibiting aminopeptidases with Bestatin also reduced myosin content. LCM treatment decreased basal and ATP-related mitochondrial respiration, caused a transient rise in reactive oxygen species (ROS), and increased 4-Hydroxynonenal (4-HNE) in both cytosolic and mitochondrial fractions of cell lysates. COX-2 inhibition did not spare myosin content in LCM-treated myotubes, but did alter mitochondrial respiration and cytosolic oxidant levels. Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines, signaling via aminopeptidases to help maintain muscle mass. We elucidated how LCM alters mitochondrial electron flow, and increases oxidative damage by ROS and 4-HNE. Although successful in clinical trials, COX-2 inhibitors do not appear to spare muscle mass by directly working on skeletal muscle, but did alter mitochondrial function.

Nutrient deprived diets are major contributors to the development of childhood obesity and metabolic diseases. Total and site-specific adiposity, such as visceral and skeletal muscle fat, have been associated with an increased risk of metabolic syndrome, insulin resistance, and other cardiometabolic risk factors in youths. C-reactive protein (CRP), a circulating inflammatory biomarker, is an established risk factor for cardiovascular disease and is associated with adiposity even at a young age. Diet quality indexes have been developed for use in adolescents and have evaluated the relationships between diet quality and selected health outcomes. Studies that assess relationships between diet quality, adiposity measured using direct methods, and metabolic risk are lacking in youth, particularly Hispanic Americans. Therefore, the objective of this dissertation was to evaluate the relationships between diet quality, assessed by the Youth Healthy Eating Index (YHEI), measures of total and site-specific adiposity, by dual-energy x-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT), and inflammation, assessed by high-sensitivity CRP (hs-CRP) in a cross-sectional study at baseline of 576 healthy Hispanic American and non-Hispanic girls aged 8-13 years. Diet was assessed using the validated semi-quantitative Harvard Youth/Adolescent Food Frequency Questionnaire (YAQ). Diet quality was assessed from the YHEI, developed based on the YAQ. Muscle density (mg/cm³), a surrogate for fat infiltration, was measured at tibial and femoral sites using pQCT. Total body and android fat (surrogate for visceral fat) were measured by DXA. Serum hs-CRP concentrations were measured by nephelometry. Multiple linear and logistic regression analyses were employed to assess these relationships. Total YHEI score was inversely associated with total body fat percent (p= 0.01) and android percent fat (p= 0.02), but not body mass index (BMI) or muscle density. Lower "margarine and butter use" and higher "meat ratio" were associated with higher leg muscle density. Higher "meat ratio" was inversely associated with BMI and greater "multivitamin use" was inversely associated with visceral adiposity. In a subsample of 113 Hispanic girls, over 50% of study participants demonstrated non-detectable serum hs-CRP levels. In adjusted models, there was no significant relationship between overall YHEI score and hs-CRP in this sample of adolescent girls. For every 1-unit increase in "whole grains" score there was a 44% increase in odds of being in the "high" category compared to the "undetectable" category of hs-CRP, after adjusting for maturity offset, PYPAQ score, total energy intake, total body fat, and all other individual YHEI components (OR: 1.44, 95% CI: 1.06, 1.97). The odds of being in the "high" category of hs-CRP were 38% higher compared to those with "non-detectable" hs-CRP for every 1-kg/m² increase in BMI (OR: 1.33, 95% CI 1.16, 1.53, p<0.0001). The odds of being in the "high" hs-CRP category increased with every 5 percent unit increase in total body percent fat (OR: 2.38, 95% CI 1.58, 3.58, p<0.0001) and android percent fat (OR: 1.89, 95% CI 1.39, 2.57, p<0.0001), compared to those with "non-detectable" concentrations. Calf muscle density was associated with lower odds of elevated hs-CRP compared to the "non-detectable" group (OR: 0.58, 95% CI 0.35, 0.75, p<0.001). The findings suggest that higher diet quality scores are associated with lower total and visceral body fat in adolescent girls. While greater total and regional adiposity are associated with increased inflammation, independent of biological and lifestyle factors, diet quality is not significantly associated with inflammation, as assessed by concentrations of hs-CRP, in Hispanic American girls.

Having an in vivo model of development that develops quickly and efficiently is important for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; hence a compliant in vivo model would provide a pivotal foundation for deciphering muscle disease mechanisms as well as the development of myopathy-related therapeutics. Here, we take advantage of a relatively quick, cost effective, and molecularly pliable developmental model system in the Xenopus laevis (frog) embryo and establish it as an in vivo model to study the roles of sarcomeric proteins during de novo myofibrillogenesis.Using the Xenopus model, we elucidated the functional roles of Leiomodin3 (Lmod3) and Tropomodulin 4 (Tmod4) during de novo skeletal myofibrillogenesis. Tmods have been demonstrated to contribute to thin filament length uniformity by regulating both elongation and depolymerization of actin-thin filaments' pointed-ends. Lmods, which are structurally related to Tmod proteins also localize to actin filament pointed-ends. In situ hybridization studies demonstrated that of their respective families, only tmod4 and lmod3 transcripts were expressed at high levels in skeletal muscle from the earliest stages of development. When reducing their protein levels via morpholino (MO) treatment, thin filament regulation and sarcomere assembly were compromised. Surprisingly, alternate rescues (i.e., lmod3 mRNA co-injected with Tmod4 MO and vice versa) partially restored myofibril structure and actin-thin filament organization. Thus, our results not only indicate that both Tmod4 and Lmod3 are critical for myofibrillogenesis during Xenopus skeletal muscle development, but also revealed that they may share redundant functions during skeletal muscle thin filament assembly.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

Type II diabetes mellitus (T2DM) has been associated with the onset of diet-induced obesity, which is currently on the rise worldwide. T2DM is typically characterized by insulin resistance in peripheral tissues such as adipose tissue, liver, and skeletal muscle. In skeletal muscle it is widely accepted that the defective insulin action is due to the inability of the cell to sufficiently activate the insulin signalling pathway and promote systemic glucose uptake. The sarcolipin-null (KO) mouse is a potential novel model for diet-induced obesity and diabetes. KO mice become significantly more obese and display a greater glucose intolerance than wildtype (WT) mice following an 8-week high-fat diet (HFD; 42% calories from fat) but the underlying mechanisms are still unknown. In this study the role of defective skeletal muscle insulin signalling in the development of the impaired glucose tolerance in KO mice was investigated. It was hypothesized that the HFD fed KO mice would exhibit greater reductions in IRS1 tyr628 and Akt ser473 phosphorylation (i.e. decreased activation of the insulin signalling pathway) than controls. Furthermore, it was believed that KO mice would display increased phosphorylation of IRS1 ser307, which is commonly associated with insulin resistance. At 16-weeks of age KO mice and littermates were subdivided into two groups and placed on either a HFD (n=30) or chow diet (n=24) for an 8-week period. Changes in body weight, glucose tolerance, and insulin tolerance were assessed pre- and post-diet period. Following the completion of the diet intervention mice were treated with an intraperitoneal injection of insulin (0.75U/kg) or vehicle solution and sacrificed for tissue collection. Epididymal/inguinal and retroperitoneal fat pads were removed for assessment of whole body adiposity. Whole gastrocnemius muscle was excised and homogenized for Western blot analysis of several key proteins of the insulin signalling cascade. Following completion of the HFD KO mice (48.6 ± 1.6 g) weighed significantly more than HFD fed wildtype (WT) mice (41.5 ± 1.6 g), and all chow fed mice (KO: 36.8 ± 1.5 g; WT: 35.2 ± 1.2 g; p<0.001). Glucose tolerance testing showed that KO mice exhibited significantly greater glucose intolerance compared to control mice post-HFD (p<0.001). Insulin tolerance testing, however, revealed no change in insulin sensitivity in KO or WT mice post-HFD (p>0.05). The HFD fed KO mice (0.73 ± 0.06 g) had an elevated retroperitoneal fat pad weight than HFD fed WT (0.49 ± 0.05 g) and all chow fed mice (KO: 0.28 ± 0.04 g; WT: 0.24 ± 0.04 g; p<0.01). Western blot analysis revealed a similar reduction in insulin receptor substrate-1 (IRS1) tyr628 phosphorylation in both KO and WT mice following the HFD (Con WT: 2.82 ± 0.69; Con KO: 3.06 ± 0.73; HFD WT: 1.71 ± 0.28; HFD KO: 1.28 ± 0.11 fold increase over non-insulin stimulated mice; p<0.02). IRS1 ser307 phosphorylation was elevated in both genotypes post-HFD (HFD WT: 2.97 ± 1.19; HFD KO: 2.17 ± 0.59 fold increase over standard chow fed control mice; p<0.03). Insulin treatment did not stimulate phosphorylation of Akt ser473 in KO or WT mice regardless of diet (p>0.05). In summary there was no difference between KO and WT mice in skeletal muscle insulin sensitivity as assessed by the phosphorylation of insulin signalling intermediates. An increase in IRS1 ser307 phosphorylation appears to be the primary mechanism for the reduced activation of IRS1 following the HFD in both KO and WT mice. However, the results from the current investigation did not support the notion that impaired skeletal muscle insulin signalling is responsible for the more pronounced diet-induced glucose intolerance observed in KO mice. Future studies investigating the viability of skeletal muscle GLUT4 translocation and glucose uptake as well as the glucose-induced insulin secretion of pancreatic β-cells following consumption of a HFD would help elucidate the mechanism of glucose intolerance in KO mice.