Skeletal muscle satellite cells (SC) are essential in muscle repair and regeneration. The role of SCs in mediating hypertrophic adaptations following resistance training has been widely studied. Recent evidence from endurance training studies suggest that SCs may also play a role in mediating non-hypertrophic adaptations. Indeed, it has been shown that satellite cells respond to endurance training. Work in rodent models suggest that exercise intensity may play an important role in expanding the SC pool whereas the results of endurance training studies in humans are much less consistent. Limited evidence also suggest that exercise intensity may be important in mediating exercised-induced capillarization following endurance training in humans. In both instances, it is unknown whether the on-and-off pattern characteristic of interval training (i.e. the rest-work cycles) plays a role in the magnitude of these skeletal muscle responses to this type of exercise. Thus, we sought to determine if the rest-to-work cycle plays a role in the scope of these skeletal muscle responses by comparing the SC response and capillarization to two distinct work-matched protocols that are performed at the same intensity but at two different exercise patterns (interval versus continuous). We hypothesized that interval exercise will elicit a greater SC response and induce greater capillary growth compared to work-matched continuous exercise. Ten young active individuals performed 12 sessions of counterweighted single-leg cycling over 4 weeks. Each leg was randomly assigned to Interval (INT) (10 x 3-min intervals at 50% of single leg peak power output (PPO), with 1 min recovery) or Continuous (CONTIN) (30 min at 50% PPO, followed by 10 min recovery), which were performed 5 min apart on each day, in an alternating order. Resting muscle biopsies were obtained from the vastus lateralis pre- and post-training. Immunofluorescent microscopy of muscle cross sections was used to assess muscle fibre capillarization, SC expansion and activation. Western blot analysis was performed on pro-angiogenic factors, specifically VEGF and VEGFR2. Mixed muscle SC activation increased in the INT leg compared to the pre-training time-point (3.2 ± 0.5 vs. 1.5 ± 0.2 Pax7+/MyoD+ cells/ 100 myofibers, respectively; P < 0.05). Mixed muscle SC activation did not increase significantly in the CONTIN leg compared to the pre-training time-point and there was no significant difference in activation between CONTIN and INT post-training. With regards to capillarization, no differences in type I or type II muscle fibre CC (capillary contacts), C/Fi (individual capillary-to-fibre ratio) or CFPE (capillary-to-fibre perimeter exchange index) were observed post-training or between legs. No significant expansion of the SC pool occurred in either legs post-training and no increases in fibre cross-sectional area was observed. This study presents novel evidence of mixed muscle SC activation following interval exercise training that is not observed following work- and intensity-matched continuous exercise training. This suggest that the rest-to-work cycle associated with interval exercise may dictate, to some extent, SC activation whereas it may not be a primary stimulus for training-induced changes in capillarization. / Thesis / Master of Science (MSc)

Satellite cells are adult stem cells that contribute to hypertrophy and repair in muscles. It is hypothesized that in muscular dystrophy, the satellite cells population is depleted at a very early age, due to repeated muscle damage and repair. Satellite cell transplantation is a potentially useful therapy for muscle diseases, but the lack of an efficient delivery system has hindered its application. The presented work focuses on two specific aims that address the need for more effective cell delivery methods for cell-based therapy. In Specific Aim 1 enhanced tissue culture techniques, such as heat stress, are used to increase cell survival in satellite cell transplantation studies. Also addressed within this specific aim are methods to label and evaluate performance using real-time PCR techniques.Although much work remains to enhancing the viability of in vitro expanded myoblasts derived from satellite cells, a second important hurdle is the systemic delivery of satellite cells to multiple sites (all muscles, in the case of muscular dystrophies). In vitro and in vivo experiments are being undertaken to explore the physiological role of cell signaling systems involved in directed migration and to determine if these chemokine and growth factors can be manipulated to enhance efficacy of cell-based therapies involving skeletal muscle satellite cells. Specific Aim 2 addresses migration of satellite cells to sites of injury and methods to track transplanted cells within the host. Presented here is the use of FAST SPECT II imaging of 111-Indium oxine radiolabeled satellite cells. The long lifetime of 111-indium oxine and the ability to quantify label using FAST SPECT imaging techniques make this technique ideal for in-vivo tracking of transplanted satellite cells for week long studies. Without in-vivo imaging techniques cell fate studies require sequential animal sacrifice with histological sectioning. This not only increases the number of animals used but also adds a significant inter-animal variability to their assessment. The determination of cell fate after transplantation will have a major impact on cell therapy for treatment of muscle disease as well as other stem cell therapies.

Young female rats treated with trenbolone demonstrated an increase in weight gain per day and overall weight increase during the treatment period. Trenbolone treated rats also experienced improved feed efficiency. Muscles removed from the lower hind limb of trenbolone treated rats had a greater DNA to protein ratio than muscles from control animals. However, there was no significant difference in wet muscle weight between trenbolone treated and control muscles. Satellite cells from untreated female rats were not responsive to trenbolone added in vitro. In studies utilizing serum free medium, trenbolone alone, and in the presence of growth factors, could not stimulate proliferation above controls. In similar serum free medium studies, satellite cells from trenbolone treated rats were more responsive to growth factors than cells from control rats.

Rotator cuff injury is a debilitating condition and when injury cannot be managed through non-operative procedures, surgery is required. To determine explanations for the failure of surgery to restore joint function, human muscle biopsies of supraspinatus were studied compared to deltoid. Histology showed atrophy and a tendency toward fibrosis/fatty infiltration in injured supraspinatus. Findings from AChR-subunit western blot and Sema3A localization around satellite cells suggest supraspinatus denervation. Nucleotide incorporation to quantify satellite cell activation in culture showed a significant increase in BrdU+(active) satellite cells in supraspinatus treated with a nitric oxide-donor drug, but not deltoid muscle. The application of principal component analysis to these data extracted components that suggest variables assaying muscle atrophy, satellite cell activity, and fibrosis contribute strongly to the observed variability. The results suggest supraspinatus muscle of the injured rotator cuff is atrophic, denervated, possibly subject to fibro-fatty infiltration, and support the idea that treatment could promote growth in atrophic supraspinatus to improve functional outcomes. / October 2015

OBJECTIVE: Tumor necrosis factor-alpha (TNF-α) is a multifunctional proinflammatory cytokine that plays a critical role in mediating inflammatory and immunological responses. TNF-α has been shown to elicit both beneficial and detrimental biological effects by acting through its two receptors, TNFR1/p55 and TNFR2/p75. Previous studies from this laboratory have shown that TNF-TNFR2/p75 signaling plays a critical role in ischemia-induced neovascularization in muscle and heart tissues. However, the role of TNF-TNFR2/p75 signaling in ischemia induced inflammation and muscle regeneration remains to be characterized. METHODS: To evaluate ischemia induced inflammation responses, young wild type (WT) and young TNFR2/p75 knockout (p75KO) mice were subjected to unilateral hind limb ischemia (HLI) surgery. Operated hind limb tissue samples were collected at 1, 3, 7, and 10 days post-HLI surgery and studied for neutrophil (myeloperoxidase-1 positive cells) and macrophage (F4/80 positive cells) infiltration as well as satellite-cell activation (neural cell adhesion molecule positive cells) at each time point. To determine possible synergistically negative roles of tissue aging and the absence of TNFR2/p75 in either the tissue or bone marrow (BM), two chimeric BM transplantation (BMT) models were generated where young Green Fluorescent Protein (GFP) positive (+) p75KO and WT BM-derived cells were transplanted into adult p75KO mice. HLI surgery was performed one month post-BMT, after confirming complete engraftment of the recipient BM with GFP donor cells. Operated hind limb tissue samples were evaluated up to 28 days post-surgery to examine proliferation and apoptosis of BM-derived cells in ischemic tissue. RESULTS: Ischemia induced significant and long-lasting inflammation associated with a considerable decrease in satellite-cell activation in p75KO muscle tissue 1-10 days post-HLI surgery. For the BMT studies, in adult p75KO with the WT-BMT, proliferative (Ki67+) cells were detected only by day 28 and were exclusively GFP (+), suggesting delayed contribution of young WT-BM cell to adult p75KO ischemic tissue recovery. No GFP (+) young p75KO BM cells survived in adult p75KO tissue. CONCLUSION: The data demonstrate that: (1) ischemia-induced recovery in skeletal muscle tissue is impaired in young p75KO mice; (2) inflammatory responses are significantly increased and long-lasting in p75KO mice; (3) in the absence of TNFR2/p75 signaling, satellite-cell activation is affected in p75KO mice; (4) during post-ischemic recovery, tissue aging combined with decreased/absent TNFR2/p75 signaling may have synergistically negative roles on survival and proliferation in the damaged tissue.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

C/EBPβ is a bZIP transcription factor known to be involved in various physiological processes, including adipogenesis, osteogenesis and liver development. Previous studies in this laboratory revealed an inhibition of myogenesis and reduced myogenic protein expression in 5-azacytidine treated mesenchymal stem cells retrovirally transduced to overexpress C/EBPβ. The goal of this thesis was to evaluate the role of C/EBPβ in myogenic differentiation by overexpression in C2C12 myoblasts and primary myoblasts. We demonstrate reduced MyoD protein expression and subsequent downregulation of myogenic proteins during differentiation following C/EBPβ overexpression. We localized C/EBPβ to the quiescent Pax7+ satellite cells associated with the muscle fiber. Upon satellite cell activation, we observed the downregulation of C/EBPβ protein expression prior to MyoD protein expression. Furthermore, the re-expression of C/EBPβ correlated with the loss of MyoD expression later in differentiation. Histological analysis of C/EBPβ-/- mice revealed smaller fibers and a reduced Pax7+ satellite cell population as compared to control animals. In this thesis, we propose that C/EBPβ is a negative regulator of skeletal muscle differentiation by inhibiting the expression of MyoD, thus impairing proper progression through the myogenic program. In addition, we propose a role for C/EBPβ in the maintenance of undifferentiatied satellite cells.

Age-related muscle atrophy and the importance of satellite cells in muscle maintenance, growth and repair led us to examine the effects of mechanical stretch, nitric oxide (NO), and age on satellite cell (SC) activation and gene expression in normal young and old mice. Baseline variables (body mass, muscle mass, fiber cross-sectional area (CSA), muscle strength, SC population, stretch activation and gene expression) were obtained from normal C57BL/6 mice at 3-, 8-, 12- and 18-months-of-age. Activation was assayed by 3H-thymidine incorporation into extensor digitorum longus (EDL) muscles isolated for culture. In a second experiment, muscle from 8- and 18-month-old mice was treated with one or more of: stretch; NO-donors (L-Arginine (LA), isosorbide dinitrate (ISDN)) and; Nω-nitro-L-Arginine methyl ester (LN). EDL muscles from 6-month-old mice required a greater stretch stimulus (20% vs. 10% length increase) than EDL from younger mice to increase SC activation. Stretch did not increase SC activation in mice older than 6 months-of-age. NO supplementation from an exogenous source (ISDN) increased SC activation by stretch in 8- but not 18-mo-old EDLs. In a third experiment, 8- and 18-month-old mice were subjected to 3 weeks of voluntary wheel running, or not. The EDL, tibialis anterior (TA), gastrocnemius (GAST) and quadriceps (QUAD) muscles were selected for analysis following sacrifice. The QUAD muscle from 8-month-old mice was the only muscle that demonstrated an exercise-induced increase in SC activation, elevated expression of neuronal nitric oxide synthase (NOS-I) and downregulation of myostatin, a gene that inhibits muscle growth. These results suggest mechanical stimulation of satellite cells and regulation of gene expression that controls muscle growth in voluntary contractile tissue is muscle-specific and age-dependent. / May 2009

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

C/EBPβ is a bZIP transcription factor known to be involved in various physiological processes, including adipogenesis, osteogenesis and liver development. Previous studies in this laboratory revealed an inhibition of myogenesis and reduced myogenic protein expression in 5-azacytidine treated mesenchymal stem cells retrovirally transduced to overexpress C/EBPβ. The goal of this thesis was to evaluate the role of C/EBPβ in myogenic differentiation by overexpression in C2C12 myoblasts and primary myoblasts. We demonstrate reduced MyoD protein expression and subsequent downregulation of myogenic proteins during differentiation following C/EBPβ overexpression. We localized C/EBPβ to the quiescent Pax7+ satellite cells associated with the muscle fiber. Upon satellite cell activation, we observed the downregulation of C/EBPβ protein expression prior to MyoD protein expression. Furthermore, the re-expression of C/EBPβ correlated with the loss of MyoD expression later in differentiation. Histological analysis of C/EBPβ-/- mice revealed smaller fibers and a reduced Pax7+ satellite cell population as compared to control animals. In this thesis, we propose that C/EBPβ is a negative regulator of skeletal muscle differentiation by inhibiting the expression of MyoD, thus impairing proper progression through the myogenic program. In addition, we propose a role for C/EBPβ in the maintenance of undifferentiatied satellite cells.