The objective of this study was to assess the potential role of extracellular, cytosolic, and membrane cholesterol in the proliferation and differentiation of bovine myoblasts. In the first experiment, myoblasts isolated from Angus or Angus crossbred steers were cultured with 2% lipoprotein deficient fetal calf serum (LPDS) or normal fetal calf serum. Culturing with LPDS did not alter the cytosolic or membrane cholesterol content, or myoblast differentiation, but inhibited myoblast proliferation, compared to culturing with normal fetal calf serum. In the second experiment, myoblasts were cultured with or without lovastatin, a selective inhibitor of cholesterol synthesis. Culturing with 5 μM lovastatin did not affect medium concentration of cholesterol, but reduced cytosolic and membrane cholesterol contents, compared to culturing with vehicle control. Culturing with 5 μM lovastatin inhibited both myoblast proliferation and differentiation. In the third experiment, myoblasts were cultured with or without methyl-βcyclodextrin (MβCD), a chemical that depletes cholesterol from cell membranes. Treating myoblasts with 10 mM MβCD for 30 minutes reduced membrane and cytosolic cholesterol contents while increasing medium cholesterol concentration. Treating with MβCD inhibited both myoblast proliferation and differentiation compared to treating with vehicle control. Overall, this study showed that lovastatin- or MβCD-induced reductions in cytosolic and membrane cholesterol contents were associated with reduced proliferation and differentiation in bovine myoblasts. These associations suggest that cytosolic cholesterol, membrane cholesterol, or both may play a role in bovine myoblast proliferation and differentiation. / Master of Science / The objective of this study was to assess the potential role of extracellular, cytosolic, and membrane cholesterol in the proliferation and differentiation of bovine myoblasts. In the first experiment, myoblasts isolated from Angus or Angus crossbred steers were cultured with 2% lipoprotein deficient fetal calf serum (LPDS) or normal fetal calf serum. Culturing with LPDS did not alter the cytosolic or membrane cholesterol content, or myoblast differentiation, but inhibited myoblast proliferation, compared to culturing with normal fetal calf serum. In the second experiment, myoblasts were cultured with or without lovastatin, a selective inhibitor of cholesterol synthesis. Culturing with 5 μM lovastatin did not affect medium concentration of cholesterol, but reduced cytosolic and membrane cholesterol contents, compared to culturing with vehicle control. Culturing with 5 μM lovastatin inhibited both myoblast proliferation and differentiation. In the third experiment, myoblasts were cultured with or without methyl-β-cyclodextrin (MβCD), a chemical that depletes cholesterol from cell membranes. Treating myoblasts with 10 mM MβCD for 30 minutes reduced membrane and cytosolic cholesterol contents while increasing medium cholesterol concentration. Treating with MβCD inhibited both myoblast proliferation and differentiation compared to treating with vehicle control. Overall, this study showed that lovastatin- or MβCD-induced reductions in cytosolic and membrane cholesterol contents were associated with reduced proliferation and differentiation in bovine myoblasts. These associations suggest that cytosolic cholesterol, membrane cholesterol, or both may play a role in bovine myoblast proliferation and differentiation.

No description available.

Thesis (Ph.D.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 151-183). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR29502

Thesis (M.Sc.)--University of Waikato, 2006. / Includes bibliographical references (leaves 113-128) Also available via the World Wide Web.

Thesis (M.Sc.)--University of Waikato, 2006. / Includes bibliographical references (leaves 89-106) Also available via the World Wide Web.

Three studies were conducted to achieve the following respective objectives: 1) to determine the cellular mechanism by which growth hormone (GH) stimulates skeletal muscle growth; 2) to identify the signaling pathways that mediate the different effects of insulin-like growth factor I (IGF-I) on skeletal muscle growth; and 3) to determine the role of a functionally unknown gene named SH3 and cysteine rich domain 3 (STAC3) in myogenesis. In the first study, the myogenic precursor cells, satellite cells, were isolated from cattle and allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. GH increased protein synthesis without affecting protein degradation in myotubes; GH had no effect on proliferation of myoblasts; GH had no effect on IGF-I mRNA expression in either myoblasts or myotubes. These data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle. In the second study, the signaling pathways mediating the effects of IGF-I on proliferation of bovine myoblasts and protein synthesis and degradation in bovine myotubes were identified by adding to the culture medium rapamycin, LY294002, and PD98059, which are specific inhibitors of the signaling molecules mTOR, AKT, and ERK, respectively. The effectiveness of these inhibitors was confirmed by Western blotting. Proliferation of bovine myoblasts was stimulated by IGF-I, and this stimulation was partially blocked by PD98059 and completely blocked by rapamycin or LY294002. Protein degradation in myotubes was inhibited by IGF-I and this inhibition was completely relieved by LY294002, but not by rapamycin or PD98059. Protein synthesis in myotubes was increased by IGF-I, and this increase was completely blocked by rapamycin, LY294002, or PD98059. These data demonstrate that IGF-I stimulates proliferation of bovine myoblasts and protein synthesis in bovine myotubes through both the PI3K/AKT and the MAPK signaling pathways and that IGF-I inhibits protein degradation in bovine myotubes through the PI3K/AKT pathway only. In the third study, the potential roles of STAC3 in myoblast proliferation, differentiation, and fusion were investigated. Overexpression of STAC3 inhibited differentiation of C2C12 cells (a murine myoblast cell line) and fusion of these cells into myotubes, whereas knockdown of STAC3 had the opposite effects. Either STAC3 overexpression or STAC3 knockdown had no effect on proliferation of C2C12 cells. Myoblasts from STAC3-deficient mouse embryos had a greater ability to fuse into myotubes than control myoblasts; the former cells also expressed more mRNAs for the myogenic regulators MyoD and myogenin and the adult myosin heavy chain protein MyHC1 than the latter. These results suggest that STAC3 inhibits myoblast differentiation and fusion. / Ph. D.

Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains v, 42 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

No description available.

Successful antiretroviral (ARV) treatment is associated with suppression of HIV viral load and the reduction of clinical disease progression. Despite marked improvements in ARV medication, side effects from long-term treatment, such as loss of muscle mass do occur. The mechanism by which ARVs affect muscle mass is unclear, however, published in vitro data suggests a negative effect on myoblast fusion during differentiation. The objective of this study was therefore to determine the effect of ARVs on processes required for successful myogenesis; these included proliferation, migration during wound repair, and differentiation. C2C12 mouse skeletal myoblasts and human primary culture skeletal (HSk) myoblasts were incubated with Zidovudine (nucleoside reverse transcriptase inhibitor-NRTI), Tenofovir (nucleotide reverse transcriptase inhibitor-NtRTI) or Ritonavir (protease inhibitor-PI) at a concentration range of 0.01 μM to 10 μM. Proliferation was determined using crystal violet and migration was analyzed using a 2D wound healing assay. The commitment of myoblasts into the myogenic lineage was assessed via the expression of the transcription factor Pax7. Differentiation was measured by assessing the fusion index of multinucleated myotubes. C2C12 myoblast proliferation was observed to increase significantly in response to Tenofovir (1 μM and 10 μM). In HSk cells however, proliferation was observed to decrease significantly in response to Tenofovir (1 μM). Zidovudine had no consistent effect on C2C12 proliferation at any dose tested, but caused a decrease in HSk myoblast proliferation (0.01 μM and 0.1 μM); however this was statistically non-significant. A small dose-dependent increase in C2C12 and HSk cell number, although not significant, was seen in response to Ritonavir. Wound closure results revealed both dose-dependent and time-dependent effects of Tenofovir and Zidovudine on human myoblast migration, with significant decreases in the rate of wound closure (4-7 hours) noted at 0.1 μM and 0.01 μM doses respectively. Zidovudine had no significant effect on migration while Ritonavir (0.01 μM) was observed to significantly increase percentage wound closure of human myoblasts, suggesting an increased ability to migrate during wound repair. Differentiation results indicated a decrease in myoblast fusion in response to all three ARVs. However only Ritonavir was shown to negatively affect myosin heavy chain expression. Further research into the exact mechanism of decreased fusion is required. To our knowledge, this study is the first to suggest that selected ARVs may significantly influence myoblast regeneration capabilities by modulating myoblast proliferation, migration, differentiation and fusion, and thereby decrease their myogenic capability. Extended human myoblast studies on differentiation could confirm this hypothesis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.