Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

INTRODUCTION: Heart failure with preserved ejection fraction (HFpEF) accounts for half of all clinical heart failure presentations, but unfortunately lacks effective therapies. Therefore, it has become more urgent to elucidate the pathophysiology underlying this disease, both by using patient data and the development of more accurate animal models. With clinical evidence suggesting that skeletal muscle abnormality is a significant factor in the development of exercise intolerance, this thesis investigates whether the salty drinking water, unilateral nephrectomy, and aldosterone (SAUNA) HFpEF mouse model also demonstrates similar skeletal muscle abnormality as seen in patients. METHODS: Eight-weeks old C57BL/6J mice were subjected to a left nephrectomy and given a mini-osmotic pump to deliver a continuous infusion of either saline (Sham) or aldosterone (HFpEF). The mice were then maintained on a standard rodent chow and a 1% sodium chloride solution. After 4 weeks, the soleus and gastrocnemius muscles were harvested. Histological analyses were performed to examine fiber composition, cross-sectional area of fiber, capillary density, and fibrosis. Quantitative PCR (qPCR) and western blot analyses were performed to examine the expression changes in mitochondrial oxidative phosphorylation, vasculature, fibrosis and inflammation. RESULTS: HFpEF mice showed significant increase in systolic and diastolic blood pressure, increased heart/tibia length ratio, increased wet/dry lung ratio, decreased bodyweight as well as decreased weight of soleus and gastrocnemius muscle relative to tibia length. In oxidative soleus muscle, histological analyses showed a reduction in the abundance of type 1 and type 2A oxidative fiber, reduced cross-sectional area of type 2A fiber, decreased capillary density and increased fibrosis. Molecular analyses showed alterations that are consistent with histological data as well as increased gene expression of inflammatory mediators. In glycolytic gastrocnemius muscle, histological analysis indicated cross-sectional area was reduced for type 2B fibers and increased in type 1 fibers, and decreased capillary density. However, no changes in fiber abundance or in fibrosis was observed. Molecular data was consistent with these findings and revealed an increased gene expression of inflammatory mediators CONCLUSION: Skeletal muscle in SAUNA HFpEF mice displayed significant abnormalities relative to their sham counterparts. These results thus support that SAUNA HFpEF mouse model is suitable and relevant to study skeletal muscle abnormalities and could contribute to the development of novel therapies for HFpEF. / 2025-06-21T00:00:00Z

Conditions that lead to reversible growth arrest (quiescence), promote the expression of a set of genes called growth arrest specific (GAS) genes. GAS genes play a crucial role in initiating and maintaining the entry into quiescence, while also activating stress responses to help the cell overcome the effects of the stressors. Gene profiling study examining the transcriptome has shown a vast number of genes that are upregulated during quiescence, among them is GEM1 (GTP binding protein overexpressed in skeletal muscle). GEM1 transcripts were elevated 18-fold in response to quiescence. GEM1 is a small monomeric GTPase from the Ras superfamily. It is involved in regulation of cytoskeleton reorganization, and inhibition of voltage gated calcium channels that ultimately prevents hormone secretion. A preliminary study determined that GEM1 is packaged into extracellular vesicles (EV). GEM1 is also reported to promote lipid accumulation and adipogenesis in goat pre-adipocytes. GEM1 is also reported to bind transcription factors that are involved in lipid homeostasis pathways. Thus, it is probable that GEM1 may play a major role in EV formation and/or release, and lipid homeostasis. This study examined the expression of GEM1 at the protein level and validates its candidacy as a GAS gene. We also created two GEM1-shRNA retroviral constructs capable of partially downregulating GEM1 expression which can serve as a molecular tool for further characterizing the function of GEM1 in quiescent CEF. / Thesis / Bachelor of Science (BSc) / GEM1 is a small monomeric GTPase, implicated in a variety of roles in eukaryotes. It plays a role in regulating adipogenesis, and hormone secretion. Most notably it regulates cytoskeleton reorganization in response to changes in calcium concentrations. Gene profiling done by Bédard Lab identified that GEM1 transcripts were highly elevated in reversible growth arrested chicken embryo fibroblasts (CEF). In this study we further explore and characterize the protein expression of GEM1 in quiescent CEF. We also design and test shRNAi retroviral constructs to downregulate GEM1 in quiescent CEF.

Skeletal muscle is a plastic tissue capable of responding to environmental perturbations. Increased loading via resistance exercise (RE) activates muscle protein synthesis (MPS) and, to a lesser extent, muscle protein breakdown (MPB). The ingestion of protein further stimulates MPS and suppresses MPB, inducing a positive net protein balance and protein accretion – i.e., muscle hypertrophy. In contrast, muscle unloading reduces MPS, which is thought to be the key driver underpinning skeletal muscle atrophy. The degree of muscle hypertrophy and atrophy in response to loading and unloading varies significantly between individuals and provides an opportunity to investigate the molecular regulators of skeletal muscle remodelling. To that end, we developed a novel unilateral model in which one leg was subjected to RE to induce hypertrophy (Hyp) and the contralateral limb was immobilized to induce atrophy (At). In study 1, we characterized the morphological changes induced by our HypAt model and validated the use of ultrasonography to measure changes in muscle size in both limbs. We discovered that by assessing the differential change in muscle size between legs we reduced the coefficient of variation between subjects. This enabled a more in-depth means-based characterization of the molecular regulators of skeletal muscle remodelling. Indeed, we discovered significantly more genes regulated by muscle remodelling than similarly-sized studies. We also identified a transcriptional signature that scaled with lean mass gains in three independent cohorts and included RNA species that were only modulated at their untranslated regions. Finally, in study 3 we simultaneously measured MPS and MPB in response to short-term immobilization (4 days) and demonstrated for the first time that MPB is statistically unchanged by unloading. Taken together, these studies contribute significantly to our understanding of skeletal muscle remodelling under different loading states and provide a valuable hypothesis-generating resource for future research in the field. / Thesis / Doctor of Philosophy (PhD) / Adaptations of skeletal muscle to loading and unloading are variable between individuals. Herein, we employed a unilateral approach to better understand the drivers of this variability by assessing the influence of resistance training (RT) and disuse on muscle protein turnover and gene expression. First, we validated the use of ultrasound for measuring changes in muscle size in response to loading and unloading. We then identified thousands of genes regulated by loading status and discovered many that were correlated with lean mass gain – some of which would not have been detected without our model. We also demonstrated that RT-induced increases in muscle protein synthesis were not associated with changes in muscle size; however, reductions in muscle protein synthesis were associated with the degree of muscle atrophy observed in response to disuse. Together, these studies contribute significantly to our understanding of how skeletal muscle size is regulated by muscle loading and unloading.

In the past decade there has been a significant increase in focus on the effect upper body vibration (UBV) has on the recovery of skeletal muscle after exercise-induced muscle damage. Recovery can be defined and investigated using a wide variety of methods. This study used three different measurements to track muscle recovery over 7 days following an exercise muscle damage protocol and applied vibration to a mathematical model. A visual analog scale (VAS) was used to measure muscle pain, a strain gauge was used to obtain maximum voluntary isometric contraction (MVIC) strength measurements, and shear wave elastography (SWE) represented muscle stiffness over the 7-day experiment. Thirty-three participants were divided into three groups. The first was a control group (C) that experienced no exercise and no therapy. The no vibration group (NV) performed the damage an exercise protocol but received no therapy. The vibration group (V) performed the same exercise protocol but also received vibration therapy. The exercise protocol consisted of 100 dumbbell curls at starting at 50% of their MVIC with one minute of rest after each set of ten. The data provided convincing evidence (27.2%, p < 0.0001) that group NV was not back to its normal stiffness after 7 days unlike group V, which was shown not to be any different from its baseline at the end of the week (9.15%, p = 0.137). Three vibration factors (����1, ����2, ����3) were added to a skeletal muscle regeneration model (SK) to simulate how vibration affects muscle regeneration. The three factors were determined by analyzing previous research to understand how vibration affects cells in the regeneration process. Adding these into SK decreased the time to recovery from about 13 days to about 7 days. Recovery was defined by reaching 10% of the original number of myofibers within the damaged muscle.

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