Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vii, 78 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 52-62).

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains ix, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 88-104).

Efficient muscle regeneration is essential in mammals in order to overcome daily stress such as wounds, exercise and pathologic processes. This regeneration relies on muscle stem cells, the satellite cells. After a lesion, satellite cells are activated, proliferate and differentiate in fonctionnal muscle fibers. Our laboratory has previously shown that the transcription factor GATA-6 is expressed in the satellite cells. The present thesis confirms the expression of this factor in this cell type. Also, it seems that GATA-6 could be implicated in the maintaining of quiescence of these cells. The GATA-6 heterozygous mouse muscle is characterized by an increase level of Myf5 and Pax7+ cells. Moreover, suppression of one copy of the GATA-6 gene in a muscular dystrophy model mouse, the mdx mice, alleviates its phenotype. Further experiments on a muscle-specific GATA-6 null mouse will allow a better understanding of the role of GATA-6 in muscle regeneration. / Keywords. GATA-6, muscle regeneration, mdx, 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.

Satellite cells are muscle stem cells that are responsible for the growth and repair of skeletal muscle tissue. Satellite cells typically exist in a quiescent state in their niche between the sarcolemma and basal lamina. In response to muscle tissue injury, activated satellite cells, otherwise known as myoblasts, migrate to the site of injury where they proliferate and subsequently differentiate and fuse to repair damaged myofibers. The success of muscle growth and repair is highly dependent on the speed and degree to which these myoblasts migrate, proliferate and differentiate. This overall process, referred to as myogenesis, is largely controlled by the myogenic regulatory factors, a group of basic helixloop- helix transcription factors including MyoD, Myf5, myogenin and Mrf4. It has recently been found that the Wnt family of secreted signalling proteins are highly involved in the regulation of developmental processes such as myogenesis. Wnt proteins are a family of 21 highly-conserved, secreted, cysteine-rich signalling molecules which are found in all multi-cellular organisms. Wnt signalling is highly versatile and is initiated by the binding of extracellular Wnt to cell-surface Frizzled receptors (Fz). It is highly dependent on both the Wnt isoform and Fz type and may initiate one of three known signalling pathways. Wnt3A and Wnt7A are of particular interest as they have previously been linked with myogenesis. C2C12 myoblasts over-expressing Wnt3A have been seen to have reduced levels of motility and terminal differentiation. Wnt7A is suspected to maintain a healthy satellite cell pool by regulating self-renewal; injection of recombinant Wnt7A into mouse leg muscle resulted in increased satellite cell numbers. In vitro Wnt studies have typically involved the treatment of mouse cells with conditioned medium containing Wnt, often at unknown concentrations. In our study we wished to test the effects of known concentrations of recombinant Wnt3A and Wnt7A on mouse C2C12 and donor-derived human skeletal muscle myoblasts (HSkM) in vitro. Wnt3A and Wnt7A were seen to increase the rate of C2C12 migration in a dose dependent manner. HSkM cells treated with 10 ng/ml Wnt3A also displayed increased motility. Neither Wnt3A nor Wnt7A were seen to have any significant effects on the proliferation of C2C12 or HSkM cells. Wnt3A (10ng/ml and 100 ng/ml) but not Wnt7A was seen to decrease C2C12 terminal differentiation as measured by expression of myosin heavy chain (MyHC). Subsequent confocal microscopy revealed that Wnt3A significantly reduced the percentage of MyoD+ C2C12 nuclei during differentiation. A reduction in nuclear MyoD would support the observed impaired commitment to differentiation. However, donor-derived human skeletal muscle myoblasts treated with 10 ng/ml Wnt3A were not seen to have significantly reduced nuclear MyoD levels or terminal differentiation; the reason for this is unclear but may relate to a number of factors including the concentration of Wnt, Fz and co-receptor profiles and the presence of specific extracellular matrix and serum factors. These studies provide new insight into the role of Wnts in myogenesis and lay the foundation for future work on Wnt3A and Wnt7A. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2014.

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.

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.

Satellite cells are known as the definitive muscle stem cells and are responsible for skeletal muscle maintenance and repair. The capacity of these satellite cells to participate in myogenesis decreases with age and as a result, muscle repair and maintenance in an aging organism is characterized by fibrosis, lipid accumulation and atrophy, a process known as sarcopenia. Recent parabiotic studies have shown that satellite cells with reduced myogenic capability in aging muscle can be rejuvenated to undergo effective myogenesis when exposed to a young environment. Further analysis has suggested that the Wnt family of signaling proteins identified in serum is pivotal in regulating cell fate, proliferation and differentiation, during aging. Wnt3a is known to regulate fibrogenensis, Wnt10b adipogenesis and Wnt7 myogenesis. In the current study, we aim to determine the cytosolic and secreted expression profiles of the three Wnt isoforms, Wnt3a, 7 and 10b, during myogenesis of early and late passage C2C12 myoblasts. We then extend our analysis to determine whether conditioned media could improve the myogenic capacity of late passage cells. Late passage C2C12 cells had elevated Wnt3a cytosolic levels along with reduced differentiation capacity and a rapidly declining Wnt7 levels, in comparison to early passage cells. The elevated Wnt3a suggests an elevated fibrogenic predisposition, whereas the declining Wnt7 cytosolic levels, a decrease in myogenic capacity. Furthermore, analysis of the secreted vs. cytosolic ratio in Wnt7 levels revealed a more rapid decline in late vs. early passage cells during differentiation, supporting the observed decreased myogenic ability. Moreover, late passage cells also showed lower Wnt10b levels compared to early passage cells. This low level of Wnt10b is likely associated with an increase in adipogenic predisposition. The results obtained in the cross-over experiments indicated that conditioned media from early passage cells did not improve the differentiation of late passage cells by the low levels of Myogenin and MHC. However, early passage cells treated with conditioned media from late passage cells surprisingly showed a marginal increase in both Myogenin and MHC levels. Interestingly, cytosolic Wnt3a and 7 in late passage cells treated with ‘young media’ were increased compared to control whereas early passage cells treated with ‘old’ media showed significantly decreased levels of Wnt3a and 7. Furthermore, early passage cells acquired a declining expression when treated with ‘young’ media whereas late passage cells had an increasing level when treated with ‘old’ media. This indicates a possible improvement in differentiation in late passage cells. Taken together, our results support a role for Wnt7 and Wnt10b in promoting myogenesis while Wnt3a may decrease myogenesis. With the increase in passage numbers, the reduced myogenic predisposition is regulated by reduced Wnt10b, 7 and elevated Wnt3a levels, respectively. Moreover, we speculate that the lack of myogenic improvement in the cross-over experiment could be the presence of unknown secreted factors in ‘young’ media that impedes myogenesis. Finally, cell lines are known to be biologically different to primary myoblasts through the accumulation of mutations which could render the cells less sensitive to growth factors. Therefore, it is imperative that the current study is repeated with primary culture myoblasts. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2006" Includes bibliographical references.