Background: Heart transplantation remains the gold standard for end-stage heart failure, with current ex vivo organ storage times limited to 4 to 6 h before critical tissue damage occurs. Many preservation solutions exist in an attempt to limit both ischemic and reperfusion damage. In order to compare the effects of various storage solutions, mitochondrial function can be used to provide a sensitive analysis of cellular metabolic function. Methods: Experimental plates were seeded with cardiac myoblasts and kept in suspended animation for either 4 or 8 h at either 4(o) or 21 degrees C, in Celsior (R), Perfadex (R), or Somah storage solutions. Cells were then reanimated for 1 h at 37 degrees C to simulate a reperfusion or clinical transplant scenario. Cellular bioenergetics were measured immediately thereafter to examine biochemical differences between preservation solutions and their effectiveness on preserving metabolic function. Results: The oxygen consumption rates of Somah solution were significantly higher than Celsior (R) and Perfadex (R) at 4 degrees C, with the exception of Perfadex (R) at 4(o) for 4 h. This effect was sustained up to 8 h. At 21 degrees C, oxygen consumption rates of Somah solution are significantly higher than Celsior (R) and Perfadex (R) at basal conditions after 4 h, but this effect is not sustained after 8 h. Conclusions: The purpose of this experiment was to study the efficacy of various preservation solutions on a mitochondrial level. The significantly higher oxygen consumption rates of Somah at 4 degrees C suggests that Somah solution may have the ability to protect cellular mitochondrial integrity, improve transplanted organ function by reducing ischemic-reperfusion injury, and thereby improve transplant outcomes. Given that Somah offers benefits over Celsior (R) and Perfadex (R) at 4 degrees C, it should be a target in future organ preservation solution research.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 138-149).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-109).

Thesis (Ph. D.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 153-184). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ67888.

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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.

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 (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 61-67). Also available on the World Wide Web.

Thesis (Ph.D.)--York University, 2009. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 275-298). 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:NR51670

The transcription factor Six1 of the sine oculis homeobox family has been tied to skeletal muscle formation. Work completed thus far has allowed our research team to identify the precise mechanism by which Six1 regulates the expression of MyoD, a key myogenic gene, in muscle stem cells. Furthermore, loss-of-function of this protein, mediated by RNA interference, has implicated Six1 as essential towards normal myogenic differentiation. However, beyond Six1 and its involvement towards myogenesis, our data also suggests the transcription factor as a potential regulator of the cell cycle. Data from our lab shows that loss of Six1 expression significantly impairs primary myoblast proliferation and appears to impair satellite cell activation in response to muscle injury in vivo. Furthermore, loss of Six1 decreases the expression of key cell cycle genes. Combining functional genomics approaches such as ChIP-Seq and Gene Expression Profiling together with Gene Ontology Term Enrichment shows a significant representation for biological processes regarding the cell cycle and its regulation; these biological clusters contain a large subset of genes that are bound and modulated by Six1. In particular, Ccnd1 was found to display a similar expression pattern as Six1 in growing myoblasts and its expression was found to be directly controlled by Six1. Furthermore, Ccnd1 over-expression was sufficient to rescue the Six1-knockdown associated cell cycle phenotype. Together, these data suggest that in response to injury Six1 enhances the expression of the cell cycle gene Ccnd1 thus modulating myoblast proliferation for muscle regeneration.