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<p>Adult skeletal muscle has the remarkable capacity of regenerating in response to stressors, such as overuse, injury, or myopathic conditions. A fundamental contributor to the regenerative process is satellite cells, which are the primary stem cells of skeletal muscle. Uncovering factors involved in satellite cell function will greatly improve their therapeutic potential, especially for patients suffering from myopathic diseases.</p> <p>The protein Xin was previously identified as being highly upregulated in damaged skeletal muscle and localized to the satellite cell population, however its purpose there has not been elucidated. Therefore the overall goal of this study was to determine the role of Xin during skeletal muscle regeneration and within its resident stem cell population. This was approached using Xin knockdown (Xin shRNA) and knockout (Xin-/- mice) models, whereby any deficits or changes in the regenerative process can be attributed to the lack/absence of Xin. The results of the following studies reveal that when Xin expression is reduced or absent, muscle regeneration is impaired, satellite cell activation is altered, and muscle fiber morphology moves towards a myopathic state.</p> <p>Furthermore, since Xin has been shown to be upregulated during regeneration, it was interesting to study the expression of Xin in human myopathic muscle which is in a constant state of regeneration. It was observed that Xin expression correlates with degree of damage in myopathic muscle, regardless of disease diagnosis. Therefore, these data have improved our understanding of muscle regeneration, satellite cell function, and suggest a clinical marker for defining muscle damage severity.</p> / Master of Science (MSc)

Duchenne muscular dystrophy (DMD) is a fatal, x-linked disease that causes progressive muscle weakness and susceptibility to damage. DMD is caused by a lack of dystrophin, a large muscle protein that performs both structural and signaling functions. A promising treatment currently in clinical trials is microdystrophin gene therapy, which delivers a truncated version of dystrophin to muscle via a viral vector. Preclinical studies have established efficacy of microdystrophin to improve muscle quality and function. With clinical success of this treatment, patients affected by DMD could become more physically active. However, the effect of exercise on both dystrophic and gene therapy-treated muscles is unclear. Recently, we demonstrated that microdystrophin gene therapy with and without 21 weeks of voluntary wheel running (VWR) improved treadmill time to fatigue and in vivo plantarflexor torque output in young mdx mice, a mouse model of DMD. Although treated mice could run well, diaphragm force and power output were blunted by VWR. A subsequent study tested longevity of two different microdystrophin gene therapy constructs in combination with VWR. Versions of each construct are being tested in clinical trials. Construct 1 contained the nNOS-binding site found in full-length dystrophin, which localizes nNOS to the sarcolemma and reduces functional ischemia of exercising limb muscles, while construct 2 lacked the nNOS-binding site and was the same microdystrophin used in the previous study. Gene- therapy treated mice that were sedentary or performed 52 weeks of VWR demonstrated similar outcomes including increased plantarflexor torque and exceptional treadmill endurance capacity. However, ex vivo diaphragm and soleus force, as well as metabolic enzyme and mitochondrial respiration assays were differentially improved, revealing unique physiological adaptations to each microdystrophin construct. Together, the data demonstrated that response to exercise after gene therapy treatment was variable and dependent on age, microdystrophin construct, and muscle type. / Doctor of Philosophy / Duchenne muscular dystrophy (DMD) is a rare, fatal muscle disease that causes progressive muscle weakness and cardiorespiratory failure. Available treatments, such as corticosteroids, slow progression of the disease but do not address the underlying genetic cause. DMD is caused by a genetic mutation that results in the loss of the muscle protein dystrophin. Microdystrophin gene therapy aims to address the genetic cause of the disease by using a non-pathogenic virus to deliver microdystrophin, a small, functional version of dystrophin, to muscle. This gene therapy is in clinical trials, and, if it is successful, treated patients will likely want to engage in more physical activity than previously possible due to muscle weakness. However, the effects of physical activity on muscles treated with gene therapy are unclear. Therefore, we conducted two studies to test the effects of voluntary wheel running on microdystrophin gene therapy in the mdx mouse, a model of DMD. The first study demonstrated that voluntary wheel running was beneficial to whole-body muscle function in mice treated with microdystrophin gene therapy. However, adaptations to the gene therapy and voluntary wheel running were variable in individual muscles. In the second study, we tested two microdystrophin constructs, which each contain different structural components of full-length dystrophin. In addition, mice ran for 52 weeks, more than twice as long as the first study. The results of the second study found that adaptations in individual muscles depended on microdystrophin construct and activity level. Additionally, we confirmed that voluntary wheel running was beneficial to whole-body function of microdystrophin–treated muscles. Together, these studies demonstrated that adaptations of gene therapy-treated muscles were dependent on microdystrophin structure, activity level, and age.

The main goal for my research was to challenge the long held belief that the mechanical properties of maturing dystrophic compared to control skeletal muscle membranes are weaker, leading to onset of Duchenne muscular dystrophy (DMD). We built on a previous report from our lab that suggested sarcolemmal membranes from dystrophic mice are not more susceptible to damage early in maturation (i.e., age 9-12 days) and determined if and when muscle mechanical properties change as the mice mature. Across four studies, I have helped define the role of dystrophin-deficient skeletal muscle membranes in the onset of DMD. A linear viscoelastic muscle model was used to determine passive stiffness and damping in control and dystrophic muscles from maturing mice aged 14-35 days. Results confirmed my hypothesis that there are no differences in passive mechanical properties between normal and dystrophic mice. Recognizing the limitations of the linear model, a nonlinear model was developed to determine the stiffness and damping of active and passive dystrophic muscles from maturing mice aged 21 and 35 days. The nonlinear model achieved a significantly better fit to experimental data than the linear model when muscles were stretched to 15% strain beyond resting length. Active and passive mechanical properties of dystrophic mice were not different than control at 14 and 28 days of age. The previously developed nonlinear model was used to determine a more complete time-course (14-100 days of age) of dystrophic muscle mechanical properties. There was no difference in passive stiffness between mdx and control muscles at each age. However, the mdx:utrn-/- muscles showed increased stiffness compared to control and mdx muscles at 21 and 28 days, suggesting a temporary change within the muscle that only occurs with a lack of both utrophin and dystrophin. Fast-twitch and slow-twitch muscle mechanical properties were compared in control and dystrophic mice aged 3, 5, and 9 weeks of age. Dystrophic and control slow-twitch muscles did not have different mechanical properties, suggesting that a lack of dystrophin does not affect slow-twitch muscles during maturation (3-5 weeks) or well after maturation (9 weeks). / Ph. D.

Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients. / Ph. D.