Thesis (Ph. D.)--Washington State University, May 2006. / Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Jane A. Boles. Includes bibliographical references (leaves 59-68).

Resistance exercise training is the most effective and accepted strategy for increasing skeletal muscle mass and strength. There is tremendous individual variability in the adaptive response to exercise and the source(s) contributing to this variability are largely unknown. Recent evidence in the literature supports the notion that capillaries may be a potential target for improving outcomes to chronic resistance exercise. Aerobic exercise training is a proven stimulus for eliciting angiogenesis and increasing capillary content. Therefore, we hypothesize that completing a period of aerobic training prior to resistance training will result in a greater increase in fibre cross sectional area (CSA) compared to resistance training alone. Fourteen participants (8M, 6F) completed 6 weeks of unilateral single leg aerobic training prior to undergoing 10 weeks of bilateral lower body resistance exercise training. Performance and anthropometric measures were completed at baseline, post aerobic training and post resistance training. Skeletal muscle biopsies were obtained from the vastus lateralis and immunofluorescent staining of muscle cross sections was completed to determine fibre CSA and satellite cell content. Following unilateral aerobic training, single leg VO2 work peak (Watts) (p<0.001), and oxygen consumption (O2 mL  min-1) (p=.0033) was significantly higher in the aerobically trained limb (EX) versus the control (CTL) limb. Capillary to perimeter fibre exchange index (CFPE) (p<0.05), a measure of microvascular perfusion, was significantly higher in the EX versus CTL limb following unilateral aerobic training. Resistance training resulted in increases in 1-repetition maximum of both squat (p<0.0001) and leg press (p<0.0001). A main effect of time was observed for limb fat free mass (p<0.0001) as determined via DEXA. Type-II fibre CSA of the EX limb was greater (p<0.05) versus CTL limb following resistance exercise training. Type-II fibre associated satellite cell content of the CTL limb was elevated (p<0.01) following resistance training. Results suggest that a period of unilateral aerobic training elevates the aerobic capacity and relative microvascular perfusion of the trained leg significantly in comparison to the non-aerobically conditioned limb. Subsequent resistance training, bilateral leg strength increased post resistance training while type II CSA increased in the aerobically pre-conditioned limb following resistance training. Collectively, these results suggest that a period of aerobic preconditioning may augment the muscle’s ability to respond to a hypertrophic stimulus. / Thesis / Master of Science (MSc) / Resistance exercise training is the most effective and accepted strategy for increasing skeletal muscle mass and strength. Yet, there is tremendous individual variability in the adaptive response to exercise and the source(s) contributing to this variability are largely unknown. Recently, evidence has emerged suggesting that capillaries may be a potential target for enhancing the adaptive response to chronic resistance exercise training. Research has only begun to characterize the extent to which microvascular perfusion (capillarization and blood flow to the muscle) plays a role in muscle health and resistance training outcomes. Currently, it is unknown if elevating microvascular perfusion is enough to facilitate greater accretion (hypertrophy) of muscle mass and strength following resistance training. Therefore, the current study hypothesized that increased microvascular perfusion induced by a pre-conditioning period of aerobic training, lasting 6-weeks, would be sufficient to enhance muscle accretion (hypertrophy) and elevate muscle stem cell content following resistance exercise training. To examine this, a cohort of young men and women performed 6 weeks of unilateral (single-leg) cycling following by 10 weeks of bilateral (both legs) resistance exercise training. Results demonstrated an increased oxidative capacity and capillary perfusion in the aerobically-trained limb following single-leg cycling, as expected. Consistent with our initial hypothesis, we observed superior muscle hypertrophy of type-II muscle fibres (increased fibre cross-sectional area), in the aerobically-conditioned limb following resistance training. The results suggest that muscle capillarization may be a determinant and facilitator of adaptation to resistance training and its outcomes.

Pax7 is a paired box transcription factor expressed by all satellite cells which are critically required for muscle regeneration and growth. The absolute requirements of Pax7 in the maintenance of the satellite cell pool are widely acknowledged. However the mechanisms by which Pax7 executes muscle regeneration or contributes to satellite cell homeostasis remain elusive. We performed cell and molecular analysis of Pax7 null satellite cells to investigate muscle stem cell function. Through genome wide studies, we found that genes involved in cell cell interactions, regulation of migration, control of lipid metabolism and inhibition of myogenic differentiation were significantly perturbed in Pax7 null satellite cells. Analysis of satellite cells in vitro showed that Pax7 null satellite cells undergo precocious myogenic differentiation and have perturbed expression of genes involved in the Notch signaling pathway. We showed that Notch 1 is a novel Pax7 target gene and by using a genetic approach we demonstrated that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) in Pax7 null satellite cells is sufficient to maintain the satellite cell pool as well as to restore their proliferation. Instead of differentiating into myogenic cells and in the absence of a myogenic cue, NICD1 Pax7 null satellite cells become a source of ectopic brown fat within muscles and give rise to brown adipocytes both in vivo and in vitro. In conclusion we showed that Notch 1 partially rescues Pax7 deficient satellite cells loss and proliferation. Additionally we provide the first evidence that Notch signalling contributes to satellite cell fate by inhibiting terminal myogenic differentiation and inducing brown adipogenesis.

Skeletal muscle has the remarkable ability to remodel and repair when exposed to various stimuli such as exercise training and injury. Many factors contribute to the maintenance of healthy muscle mass throughout the lifespan including a functional population of resident muscle stem cells, commonly referred to as satellite cells (SC). When SC become active in response to a stimulus they proliferate and differentiate, eventually fusing to existing myofibres or to each other giving rise to new myotubes; while some SC revert to quiescence to maintain the SC pool. This process is termed 'the myogenic programme' and is governed by a complex network of transcription factors termed myogenic regulatory factors. SC are absolutely necessary for the repair of skeletal muscle, however, their role in mediating skeletal muscle remodelling following exercise training remains debatable. The effect of resistance exercise on SC content has been extensively studied in humans. However, a paucity of information exists in regards to the effect of 'non-resistance' type exercise training on SC content and function in healthy young adults. The purpose of this thesis was to determine the impact of nonhypertrophic exercise training on SC content. We examined the effect of high intensity interval training on the SC pool and determined that there was an increase in SC associated with remodelling hybrid fibres. We extended upon these findings by demonstrating that several high intensity interval training paradigms and traditional endurance training all resulted in an increase in SC pool activity without an overall expansion of the SC pool. Skeletal muscle regeneration is impaired in old rodents and is associated with a reduction in SC content. We therefore sought to determine whether nonhypertrophic exercise training in old mice was able to improve the regenerative response following injury. Exercise training resulted in an increase in SC content and improved skeletal muscle regeneration in old mice. In addition to previous work implicating SC in mediating skeletal muscle hypertrophy induced by resistance exercise training, this thesis provides evidence that SC are able to respond to exercise stimuli that are nonhypertrophic in nature. In addition, we demonstrate that nonhypertrophic exercise training results in an increase in SC content in old mice and this likely contributed to the improvement in skeletal muscle regeneration observed in old mice pre-conditioned with regular exercise. / Thesis / Doctor of Philosophy (PhD)

This study examined the effects of basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-β) on the proliferation and differentiation of bovine satellite cells (BSC) in vitro. Cells were treated with serum-free defined media containing varying concentrations of bFGF, IGF-I and TGF-β. On day 3 of treatment total cell nuclei and myotube nuclei were determined. bFGF stimulated BSC proliferation in a dose-dependent fashion with half-maximal stimulation observed at a concentration of 5 ng/ml (p < .05). Similar results were found for IGF-I and TGF-β in the presence of FGF, with half-maximal stimulation observed at 5 ng/ml and 1 ng/ml, respectively. With regard to differentiation, TGF-beta inhibited myotube formation at concentrations above 0.05 ng/ml. IGF-I stimulated myotube formation at concentrations as low as 10 ng/ml (p < .05). These results demonstrate that proliferation and differentiation of BSC in vitro are affected by growth factors, and consequently, similar effects may be found in vivo. This information may prove to be useful in future methods of manipulating muscle growth in vivo.

Myostatin, a TGF-β superfamily member, is a key negative regulator of embryonic and postnatal muscle growth. In order to further elucidate the role of myostatin during postnatal growth, several lines of investigation were undertaken in mice. Analysis of myostatin downstream target genes identified several known and unknown genes. From these, the regulation of an androgen receptor binding co-factor, ARA70, was selected for further investigation. Reverse Northern analysis on the differentially expressed cDNA library indicated an increased expression of ARA70 in myostatin-null muscles, which was later confirmed by Northern blot and semi-quantitative PCR analysis. In corroboration, treatment of myoblast cultures with exogenous myostatin resulted in the down-regulation of ARA70, confirming that myostatin is a negative regulator of ARA70 gene expression. The role of myostatin during sarcopenia, a progressive age-related loss of skeletal muscle mass and strength, was also investigated. The atrophy associated with sarcopenia is frequently correlated with insufficient muscle regeneration, resulting from an impaired propensity of satellite cells to activate and a subsequent decline in myogenesis. Myostatin is a known inhibitor of postnatal satellite cell activation and muscle regeneration, thus muscle mass and regeneration, and satellite cell behaviour were examined in young and aged myostatin-null mice. Myostatin-null mice had increased individual muscle weights, as a consequence of massive fibre hypertrophy and hyperplasia, and an increased proportion of type IIB fibres. Aging induced oxidative fibre type changes and atrophy in the wild-type muscle while no fibre type switching was observed in the myostatin-null muscle and atrophy was minimal. No decrease in satellite cell numbers was observed with aging in both genotypes; though a gradual decline in the number of activated satellite cells was noted during aging. However, both young and aged myostatin-null mice displayed increased satellite cells and activation compared to wild-type mice, suggesting a greater myogenic potential in the myostatin-null satellite cells. Consistent with this, aged myostatin-null myoblasts proliferated faster and displayed a higher fusion index during differentiation than the aged wild-type myoblasts, confirming that the reduced sarcopenia in the myostatin-null mice was due to a preserved increase in the myoblast myogenic activity. An increase in a Pax7-only myoblast population from myostatin-null muscle indicated an enhanced satellite cell self-renewal process, consistent with the increased satellite cell number observed on the myostatin-null muscle fibres. Additionally, muscle regeneration of aged myostatin-null muscle following notexin injury was accelerated, and fibre hypertrophy and type were recovered with regeneration, unlike the aged wild-type muscle. Testing the therapeutic value of a myostatin antagonist, Mstn-ant1, indicated that a short term blockade of myostatin by the antagonist significantly enhanced muscle regeneration in aged mice after injury and during sarcopenia. Antagonism of myostatin led to satellite cell activation, increased Pax7 and MyoD protein levels, and greater myoblast and macrophage cell migration culminating in enhanced muscle regeneration in the aged mice. In conclusion, the hypertrophic phenotype associated with myostatin-null mice may in part result from increased androgen receptor (AR) activity due to the up-regulation of ARA70, given that increased expression of the AR leads to hypertrophy. Additionally, the increased muscle mass in myostatin-null mice is likely to result from an augmented myogenic potential and self-renewal process. Overall, a prolonged absence of myostatin reduced sarcopenia and the associated loss of muscle regenerative capacity. Furthermore, the antagonism of myostatin displayed significant therapeutic potential in the alleviation of sarcopenia, through the restoration of the myogenic and inflammatory responses in the aged environment. Thus, the research work clearly demonstrates the role of myostatin in sarcopenia, and documents for the first time a valid therapeutic for alleviating sarcopenia.

Skeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury. / text

Traditional meat production has major sustainability and ethical concerns. Cultivated meat helps to address these concerns by reducing the need for mass animal farming. Muscle progenitor cells (MPCs) harvested from skeletal muscle are a promising cell source for cultivated meat. While various protocols have been developed for MPC isolation, which protocol is best suited for the cultivated meat industry requires further investigation. Therefore, the purpose of this thesis was to optimize the MPC isolation technique to produce a pure myogenic cell population and provide the cultivated meat industry with standardized procedures for production. For these proof-of-concept experiments, skeletal muscles harvested from the hindlimb muscles of mice were used. Cells were isolated from the harvested muscle then subjected to one of three protocols for MPC enrichment: pre-plating, ice-cold treatment (ICT), or fluorescence activated cell sorting (FACS). The pre-plating and ICT protocols resulted in impure cell populations with few MPCs after one week in culture. Therefore, FACS using two cell-surface markers, NCAM and CD34, was employed as a more specific method for MPC sorting. CD34+NCAM1- cells grew quickly, however, unwanted cell types remained following FACS. In contrast, CD34+NCAM1+ cells had a consistent small, rounded shape and slow proliferation rate. These cells remained viable in culture for several months and had high Pax7 expression, indicating they were a pure population of myogenic cells. CD34+NCAM1+ cells maintained their capacity to differentiate after culturing for an extended period, demonstrating their potential use for cultivated meat production. The results of this study provide a better understanding of the differences between previously published MPC isolation techniques. Future studies will investigate the potential for CD34+NCAM1+ cells to be grown on a larger scale. These experiments provide insight into MPC populations that may exist in livestock species and will help to streamline the early stages of cultivated meat production. / Thesis / Master of Science (MSc) / Traditional meat production is associated with numerous challenges including animal welfare concerns, human health concerns, and harmful environmental consequences. The global population is predicted to reach 9.7 billion by 2050, emphasizing the importance of alternative food sources to meet the increased food demand. Cultivated meat is a promising new protein source, with the intended purpose of providing a sustainable food source with reduced ethical concerns compared to conventional meat. While there are several challenges to overcome throughout the production process, a major consideration in the early stages of cultivated meat production is cell sourcing. Muscle cells harvested from a tissue biopsy are one proposed starting cell source which has the potential to make up most of the tissue in cultivated meat products. This thesis aimed to improve upon previously published protocols used for muscle cell isolation and provide an optimized cell population for use in cultivated meat production. The cell sorting protocol described in this thesis provides a highly efficient technique for muscle cell purification and long-term growth. The resulting cell population has many characteristics that are pertinent to cultivated meat and may advance the early stages of production.