Occurrence of obesity has steadily increased in the human population and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury, however, the mechanism underlying these changes in satellite cell (SC) biology have yet to be explored. To test the negative impacts of obesity on SC behaviors, we fed C57BL/6 mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller numbers (P < 0.001) compared to those isolated from NC mice, and this observation was confirmed (P < 0.05) by BrdU incorporation. Moreover, in vitro differentiation assays consisting of equally seeded SCs derived from NC and HFD muscles showed that HFD SCs exhibited compromised (P < 0.001) differentiation capacity compared to NC SCs. Immunocytochemical staining of cultured SCs demonstrated that the percentage of Pax7+/MyoD- (self-renewed) SC subpopulation decreased (P < 0.001) with HFD treatment group compared to the control. In single fiber explants, a higher ratio of SCs experienced apoptotic events as revealed by the expression of cleaved caspase 3 (P < 0.001). To investigate further the impact of obesity on SC quiescence and cycling properties in vivo, we used an inducible H2B-GFP mouse model to trace the turnover rate of GFP and thus cell division under normal and obese conditions. Flow cytometric analysis revealed that SCs from HFD treatment cycled faster (P < 0.001) than their NC counterparts, as reflected by the quicker loss of the GFP intensity. To test for SC muscle regenerative capacity in vivo, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC quiescence, proliferation, differentiation, and self-renewal in vitro, ex vivo and in vivo. / MS / The prevalence of obesity in the human population has steadily increased over the past decades and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. Along with the multitude of health disorders associated with obesity, in mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury. The mechanisms underlying the impairment in muscle regeneration as seen in obesity are unknown. To better understand how obesity affects skeletal muscle, we looked at satellite cells (SC). Satellite cells, or muscle stem cells, are skeletal muscle resident cells that play a vital role in muscle repair after damage. To test the negative impacts of obesity on SC behaviors, we fed mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks to obtain an obesogenic mouse model. Our first experiments involved culturing the SCs derived from the HFD and NC mouse muscles and growing them in an artificial environment. These experiments showed SCs derived from HFD mice had a decreased ability to replicate and divide compared to those isolated from NC mice. Moreover, the SCs from the HFD mice exhibited compromised capacity to form myotubes in culture, an essential part in muscle regeneration after damage. Our next set of experiments conducted looked at individual muscle fibers isolated from mouse muscle. In these experiments the SCs on the HFD muscle fibers had a higher ratio of SCs experiencing cell death in comparison to the control. To test the SC cycling properties in the living mouse we used a mouse model to trace the activity and cell division of SCs under normal and obese conditions. Using this model revealed that SCs from HFD treatment cycled faster than their control counterparts, even in the absence of notable muscle damage. To test for SC muscle regenerative capacity after muscle damage, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle (leg muscle) of the living mouse. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC behaviors and function.

The vast majority of horses engage in some form of exercise, whether it be for leisure or competition. Despite almost half of the approximately 7.2 million horses engaging in structured athletic work, very little is known about one of the most critical facets of recovery: satellite cells (SCs). Satellite cells lie adjacent to the myofiber of skeletal muscle, poised to enter the myogenic program and fuse to the nearby muscle after a damaging event. Hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) transcript abundance increased after an exhaustive bout of endurance exercise in concert with myogenic regulator factors and preceding increased SC abundance in a previous study. This suggests that SCs may participate in repair of exercise-induced muscle damage. To assess the role of HGF in this process, equine SCs (eqSCs) were isolated from the gluteus medius of mature thoroughbred geldings for activation, proliferation and differentiation assays. Activation was not accelerated by 1, 5, 10, or 25 ng/mL HGF. Instead, 25 ng/mL HGF increased the proliferation rate of eqSC via protein kinase C δ and decreased differentiation. The influence of dietary L-citrulline, an amino acid that has the potential to influence SC activity and nutrient availability by its metabolism to L-arginine, was assessed during recovery from exercise in unfit adult horses. To model submaximal exercise, horses were exercised for 1 h at an average heart rate of 116 bpm, suggested to be typical of a heavy exercise session by the National Research Council. L-citrulline decreased myogenin mRNA abundance compared to controls while exercise increased peroxisome proliferator-activated receptor gamma coactivator 1- α (PGC1α) mRNA abundance, a master regulator of energy metabolism, at 1 d post-exercise. Although SCs were not activated in response to a single bout of submaximal exercise, metabolic regulators increased in the early period of recovery. Through these studies eqSC dynamics during exercise are better defined. / Doctor of Philosophy / The horse is well-known as an athletic creature and is often used in amateur and professional athletic events. Despite its popularity as a pastime in low and high-stakes competition, certain facets directly related to performance during exercise remain relatively unstudied. One crucial component of recovery from exercise is the intrinsic ability of skeletal muscle to repair exercise-induced muscle damage. This is accomplished largely through the incorporation of new nuclei, which originate from a position orbiting the muscle, hence the name satellite cells. This cell is essential to muscle regeneration from injury as often demonstrated in rodent models, but the role of satellite cells in recovery from exercise remains elusive in all species, but particularly so in horses. For instance, whether satellite cells only contribute nuclei after exercise to stimulate gains in muscle mass or whether they may also play a role in the process of adaptation to exercise is not clearly understood. The purpose of my work was to define the response of satellite cells to hepatocyte growth factor, a factor present in skeletal muscle during exercise that is already well-studied in rodent models. Additionally, to determine whether the addition of the non-essential amino acid, citrulline, would influence satellite cells and nutrient reserves after a session of submaximal exercise. I found that hepatocyte growth factor does not influence satellite cells isolated from horses in the same way it influences those from rodents, nor through the same mechanisms. Additionally, I found that satellite cells were not stimulated after a session of submaximal exercise, but a factor involved in regulation of genetic expression that is associated with satellite cells and skeletal muscle was downregulated with the addition of citrulline. Together, these results suggest that satellite cells may behave like other species in some ways, such as some responses to hepatocyte growth factor and the lack of response to a submaximal bout of exercise, but that there is still much to be learned in order to begin to influence management and training decisions as regards skeletal muscle recovery.

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)

Satellite cells (SCs) are muscle stem cells that stay in a metabolically and mitotically quiescent state in adult skeletal muscle until activated. In mammals, SCs are activated and enter into the cell cycle for growth and regeneration. The mechanism initiating SC activation in vivo and in vitro, mediated by nitric oxide (NO) and hepatocyte growth factor (HGF) is described in the mouse model, but not in other species. Here, we assessed SC activation by counting bromodeoxyuridine (BrdU)-immuno-positive cells, and found that SC activation in zebrafish single muscle-fibre cultures is also NO and HGF dependent, peaking at 1 mM isosorbide dinitrate (ISDN, an NO donor drug) and 10 ng/mL HGF respectively, using dose-response experiments. Moreover, HGF signalling via the c-Met receptor is involved in the SC activation pathway and is considerably affected by temperature (i.e., 21 °C). Overall, understanding NO-HGF-c-Met signalling in SC activation gives new insights on fish muscle growth and conservation of regulatory pathways between species.

Satellite cells (SCs) are muscle stem cells that stay in a metabolically and mitotically quiescent state in adult skeletal muscle until activated. In mammals, SCs are activated and enter into the cell cycle for growth and regeneration. The mechanism initiating SC activation in vivo and in vitro, mediated by nitric oxide (NO) and hepatocyte growth factor (HGF) is described in the mouse model, but not in other species. Here, we assessed SC activation by counting bromodeoxyuridine (BrdU)-immuno-positive cells, and found that SC activation in zebrafish single muscle-fibre cultures is also NO and HGF dependent, peaking at 1 mM isosorbide dinitrate (ISDN, an NO donor drug) and 10 ng/mL HGF respectively, using dose-response experiments. Moreover, HGF signalling via the c-Met receptor is involved in the SC activation pathway and is considerably affected by temperature (i.e., 21 °C). Overall, understanding NO-HGF-c-Met signalling in SC activation gives new insights on fish muscle growth and conservation of regulatory pathways between species.

Intrauterine growth restriction causes impaired growth and development of mammalian fetus, and leads to long-term negative effect on postnatal growth. Among domestic animals, pigs exhibit the most severe naturally occurring IUGR and reduced postnatal muscle growth. The objectives of this research project were to: 1) determine muscle stem cell characteristics in IUGR pigs; 2) determine how intrauterine growth restriction alters protein deposition in skeletal muscle; 3) investigate whether branched-chain amino acids (BCAA) are able to enhance protein synthesis in intrauterine growth restricted (IUGR) pig muscle. Newborn piglets were considered normal body weight (NBWT) or IUGR when birth weight was within ± 0.5 SD and -2 SD of litter average respectively. Muscle satellite cell numbers, believed to be the major nuclei source for postnatal muscle growth, were lower in newborn IUGR pigs which could result in reduced muscle hypertrophy potential. In addition, cultures derived from IUGR muscle satellite cells had a lower fusion percentage. Fewer satellite cells and impaired differentiation ability may contributor to impaired muscle growth in these pigs. Protein synthesis rate was significantly lower in IUGR pig hindquarter in the first hour after feeding, but BCAA supplementation had no effect on protein synthesis in IUGR pigs. Further, eukaryotic translation initiation factor 4E (eIF4E) expression is down regulated in IUGR pig muscle. These results suggest that impaired translation initiation may provide a plausible explanation for the lower protein synthesis rates observed in IUGR pigs. Overall, reduced muscle stem cell number and changes in their activity, as well as impaired translation initiation may be important explanations for compromised postnatal muscle growth in intrauterine growth restricted pigs. / Ph. D.

<p> Local renin-angiotensin systems (RASs) have been described in many mammalian tissues. However, the role of angiotensin II (Ang II) in skeletal muscle is poorly understood with initial reports suggesting it may function to regulate overload-induced hypertrophy. Therefore, the purpose of this thesis was to 1) investigate the potential that adult skeletal muscle and muscle stem cells possess a local RAS. 2) Describe its role in regulating skeletal muscle regeneration and growth following injury and 3) demonstrate its capacity to regulate muscle stem cell activity and myogenesis. We report that cultured primary and C2C12 myoblasts and myotubes possess a local Ang II signalling system evidenced by the differential expression of angiotensinogen, angiotensin converting enzyme (ACE), and both angiotensin type 1 and 2 (AT1, AT2) receptors. Interestingly, myoblasts demonstrated the capacity to produce Ang II in spite of lacking renin expression. Furthermore, angiotensin receptors demonstrated differential localization with AT1 associated with actin filaments in proliferating myoblasts, and localized to the nucleus in differentiated myotubes. We also report that a local angiotensin system is present in vivo and responsive to myotrauma as cardiotoxin injection (to induce muscle injury) resulted in the increased staining intensity of angiotensinogen and AT1 during myogenesis with a progressive downregulation throughout the regenerative timecourse. </p> <p> To investigate the effects of Ang II signalling blockade on muscle growth and regeneration we induced muscle injury in mice supplemented with captopril (ACE inhibitor) or mice devoid of the AT1 a receptor. Histological analysis revealed that ACE inhibition resulted in a decreased muscle fibre growth, increased proportion of small myofibres, an inability to accrete myonuclei and a robust hyperplasia of muscle fibres. Similarly, AT1 a receptor ablation resulted in decreased muscle fibre growth following injury suggesting that these effects are receptor specific. </p> <p> To investigate the mechanisms underlying these effects we assessed the role of Ang II in regulating muscle satellite cell function. In vitro experiments revealed that Ang II had the ability to regulate the early response of satellite cells to muscle injury by acting as a potent transcriptional activator of quiescent myoblasts and directing their subsequent migration. Furthermore, these migratory effects were mediated through an Ang 11-induced increase in matrix metalloproteinase 2 (MMP2) content and reorganization of the actin cytoskeleton. Interestingly, Ang II may also participate in the fusion of myoblasts as captopril treatment suppressed the expression of markers of differentiation (myogenin) and maintained the expression of markers of proliferation (Pax7, Myf5). In agreement with this, IHC analysis revealed that ACE inhibition also induced a strong trend for a decrease in the proportion of myogenin positive cells following injury. Collectively, these results implicate the activation of local Ang II signalling system as a pleiotropic regulator of skeletal muscle growth. </p> / Thesis / Doctor of Philosophy (PhD)

Diabetes Mellitus is chronic lifelong condition that continues to be a global health concern. Despite the development of insulin therapy in 1921, many diabetics are likely to endure a number of co-morbidities that impact their quality of life. Today, the search for additional diabetic therapies incorporates the investigation of various organ systems for their potential in attenuating disease development. Skeletal muscle is a striated tissue that is integral to metabolism, movement, and overall wellbeing, yet its significance to Diabetes Mellitus remains understudied, as compared to other metabolic tissues. Previous work has identified that diabetes promotes adverse changes to skeletal muscle physiology, function, and morphology, contributing to a complication referred to as diabetic myopathy. The capacity to adapt to changing internal and external cues, as achieved through skeletal muscle plasticity, permits the maintenance of skeletal muscle health; a term encompassing its metabolism, function, and/or structure. This malleability is primarily regulated by the function of muscle progenitor stem cells, referred to as satellite cells. While past research has shown that satellite cells are hindered in various diabetic states, the precise mechanisms through which these observations occur remain to be elucidated. The data presented herein identify impaired satellite cell activation in two sub-types of diabetes (Pre-Diabetes and Type 1 Diabetes), and shows that such results are mediated by alterations to intrinsic signalling cascades. Additional insight into a potential unifying mechanism mediating this response led to the identification of Lipocalin-2 and its influence on satellite cell function and muscle plasticity. The results uncovered in these studies have enhanced our understanding of the response of satellite cells in diabetes, and have identified a prospective therapeutic target for the attenuation of diabetic myopathy. / Dissertation / Doctor of Philosophy (PhD)

In the equine industry, there is a need for supplements that can improve performance and muscle recovery. Tributyrin and butyrate affect satellite cells and oxidative metabolism in other species. To assess the effects of tributyrin on equine muscle repair, Thoroughbred horses were supplemented tributyrin, and a submaximal exercise test was performed. RNA isolated from the gluteal muscle of horses supplemented with tributyrin had increased myogenin mRNA. Satellite cells isolated from supplemented horses had a higher percentage of proliferating cell nuclear antigen immunopositive cells, indicating tributyrin primed satellite cells to activate. Another experiment was performed to test the effects of tributyrin supplementation on equine muscle metabolism. Horses were fed tributyrin for 30 days while partaking in light exercise training. After the supplementation period, horses performed an exhaustive exercise test. Tributyrin supplementation did not affect performance or measures of oxidative metabolism in the muscle. To measure the effects of butyrate on equine muscle metabolism, Thoroughbred horses were supplemented butyrate for 30 days. At the end of supplementation gluteal muscle from butyrate fed horses had a higher percentage of type IIA fibers. Tributyrin supplementation demonstrated positive effects on satellite cell activation, but failed to increase oxidative metabolism measures. Butyrate did statistically increase the percentage of type IIA fibers, but not oxidative enzyme activity and the modest increase seen would likely not effect performance. Higher doses and longer supplementation of butyrate and tributyrin should be investigated to see if a metabolic shift beneficial to racehorses can be achieved. Furthermore, future research should focus on tributyrin's effects on satellite cells and how supplementation can improve muscle recovery in racehorses. / Doctor of Philosophy / Thoroughbred racehorses take part in strenuous races that result in exercise induced damage to the muscle. In turn, this resulting damage to the muscle must be properly repaired before the horse can successfully race again. My projects involved finding nutritional supplements to improve equine muscle repair or metabolism. A special cell in the muscle, called a satellite cell is responsible for repairing this muscle damage. Unlike other muscle nuclei, satellite cells have the ability to divide, providing more myonuclei, able to fuse into muscle fibers for repair. In an un-damaged state, satellite cells are lying dormant in the muscle. However, upon stimulus, satellite cells leave their dormant state and become activated. Along with muscle damage, a lot of energy is used during a race. There are two main ways that energy can be made, either without oxygen (anerobic) or with oxygen (aerobic). Aerobic metabolism is able to produce more energy and is fatigue resistant. I supplemented Thoroughbred horse's diet's with two products, butyrate or tributyrin to test their effects on equine satellite cells and muscle metabolism. Ultimately, my research found that tributyrin was able to stimulate equine satellite cells activate after exercise. I also found that butyrate supplementation increased the percentage of aerobic muscle fibers.