Skeletal muscle disease (myopathy) carries an enormous psychological, social and economic impact on the lives of the patients and their caregivers. There is also an appreciable amount of economic burden on the healthcare system and our society especially when most patients are in their childhood/adolescent lives. It is not a surprising fact that a percentage of myopathies are of undetermined cause, which makes the need to identify new genes that play a critical role in muscle health of paramount importance. The Xin gene is designated as an indispensable component for the normal development and morphogenesis of striated muscle; however, the exact roles of Xin in skeletal muscle are still undefined. Studies to date have demonstrated that Xin is expressed in activated satellite cells and newly formed myofibers following tissue injury. When using in vitro reduction of Xin expression in skeletal muscle cells, the outcome was impairments in satellite cell function and muscle response to injury. That said, there is still a significant lack of knowledge in the literature regarding the in vivo effects of the absence of Xin on skeletal muscle structure and function. Therefore, the aim of our studies was to characterize skeletal muscles in the absence of Xin and identify the potential roles of Xin in maintaining muscle integrity and health. In our studies, we uncover a new form of muscle disease resulting from the lack of Xin in a mouse model (Xin-/- mice). Xin-/- skeletal muscles show a mild form of myopathy at the light microscopy level that is associated with ultrastructural and functional defects peculiar to the Ca2+ handling. In addition, Xin-/- muscle demonstrated features of mitochondrial dysfunction that are likely secondary to the Ca2+ defects caused by the absence of Xin. Our studies provide a platform to investigate the potential manifestation of “loss of function” mutation of Xin in skeletal muscle and its possibly related pathology in human diseases. / Thesis / Doctor of Philosophy (PhD) / Skeletal muscle covers the bones, produces movements and stabilizes joints. Diseases of skeletal muscle are common. Specifically, inherited diseases of skeletal muscle can cause an inability to eat, walk, run, work and even breathe. By starting at an early age, these conditions may result in the patient being bed-ridden with severe psychological, social and financial burdens for them and their caregivers. When skeletal muscles that control breathing are involved, death can occur at a young age. A considerable proportion of skeletal muscle disease are of unknown cause, and therefore, there is a constant need to discover more factors that influence muscle health to provide treatment plans for these patients. This dissertation uncovers the role of a new factor, Xin, involved in keeping skeletal muscle healthy. The achieved results of our studies will help physicians use this knowledge into identifying new types of muscle disease and providing targeted treatments for patients.

Muscle satellite cells are a distinct population of myogenic progenitors that mediate the postnatal growth and regeneration of skeletal muscle. To gain insight into the genetic regulation of satellite cell function during muscle regeneration, genes expressed specifically in these cells were identified by representational difference analysis of cDNAs. Notably, the paired-box transcription factor Pax7 was isolated as a gene specifically expressed in quiescent and activated satellite cells. Cell culture and histological analysis of PaxZ-deficient muscle revealed a complete absence of satellite cells. This result demonstrates a requirement for Pax7 upstream of MyoD and Myf5 in the specification of muscle satellite cells. Consistent with their lack of satellite cells, adult PaxT mice displayed an aggravated muscle wasting phenotype characterized by spinal kyphosis and reduced muscle mass. Acute muscle damage led to extensive calcification and deposition of adipose and fibrotic tissues with the appearance of rare regenerated myofibers. Importantly, analysis of Pax7 muscle suspensions indicated that myogenic cells expressing Pax3 and MyoD were responsible for this low level of regeneration. To characterize the role of adult stem cells in skeletal muscle, we investigated the myogenic potential of muscle-derived CD45+:Sca1+ cells in vivo during regeneration and in vitro using coculture assays. CD45+ and Sca1+ cells isolated from uninjured muscle were uniformly non-myogenic. Strikingly, 7-10% of CD45+:Sca1+ cells purified from regenerating muscle activated the myogenic program by a Pax7-dependent mechanism in response to activation of the Wnt signaling pathway. Furthermore, expression of Pax7 was sufficient to induce myogenic commitment in CD45f+Scal cells from uninjured muscle. This result demonstrates that non-satellite cell derived myogenic progenitors possess a physiological role in muscle regeneration and tissue homeostasis. Taken together, this work establishes a requirement for Pax7 in the specification of muscle satellite cells and for the myogenic recruitment of adult stem cells populations during tissue repair. Importantly, these studies also suggest that targeted therapies to activate Wnt signaling and Pax7 expression in adult stem cells will be effective for promoting muscle regeneration in patients with degenerative neuromuscular diseases or muscular dystrophies. / Thesis / Doctor of Philosophy (PhD)

Cultured meat offers a sustainable alternative to traditional meat production, addressing critical environmental and ethical issues. A key aspect of this process is the large-scale proliferation of muscle satellite cells, which can further proliferate and form the primary component of cultured meat. However, ensuring efficient cell proliferation within bioreactors is a significant challenge. Without effective and robust cell proliferation, it would be impossible to meet the production demands of cultured meat. Moreover, 3D cell spheroids tend to form tightly packed structures. As these spheroids grow larger, the limited penetration of oxygen and nutrients can lead to the formation of necrotic cores or cause cells in the central layers to experience cell cycle arrest, resulting in either irreversible senescence or reversible quiescence. This adds complexity to maintaining stable cell growth. To address these challenges, different ECM components, specifically Matrigel and Collagen I, were introduced to alter matrix stiffness and growth factor concentrations. The goal was to address issues of reduced cell proliferation and cell cycle arrest. Results demonstrated that in the absence of ECM, 3D-cultured bovine muscle satellite cells spontaneously formed myospheres but exhibited cell cycle arrest and inhibited proliferation. These issues were reversed with the addition of ECM. Increasing ECM stiffness, particularly through higher concentrations of Matrigel and Collagen I, significantly enhanced cell spreading but had a complex effect on cell proliferation. While Matrigel promoted both cell spreading and proliferation, higher stiffness and growth factor levels were associated with reduced proliferation rates, indicating a trade-off between these processes. Notably, a stiffness of 1.5 Pa with 1.56 mg/ml Matrigel yielded the highest proliferation rate, suggesting this condition might be optimal for use in bioreactor systems. Additionally, increasing matrix stiffness using Collagen I also enhanced cell spreading, indicating that cell spreading is strongly influenced by ECM stiffness. Furthermore, Matrigel reduced the expression of quiescence and senescence markers, helping to maintain cells in a proliferative state. These findings underscore the importance of optimizing ECM properties to balance cell proliferation and structural organization in 3D cultures, providing a foundation for scaling up 3D culture systems in bioreactor settings—a critical step toward large-scale cultured meat production. / Thesis / Master of Applied Science (MASc)