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.)--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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<p>The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence. </p><p>Major results of the dissertation are as follows. Culture conditions of 100 &#956;g/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.</p> / Dissertation

Myogenesis is the process of skeletal muscle tissue formation where committed muscle progenitor cells differentiate into skeletal muscle fibres. Depending on the instructive cues the muscle progenitor cells receive they will differentiate into specific fibre types with different properties. The skeletal muscle fibres can be broadly classified as fast-twitch fibres or slow-twitch fibres, based on their contractile speed. However, subgroups of fast- and slow-twitch fibres with different metabolic properties, endurance and different isoforms of sarcomeric components have also been identified, adding complexity to the process of muscle tissue patterning. The skeletal muscle tissue has the capacity to regenerate throughout life. Upon muscle tissue damage muscle satellite cells are recruited to the area of injury where they proliferate and either form new fibres similar to those damaged, or fuse with existing fibres. This thesis aims to investigate the process of muscle progenitor cell proliferation and differentiation, as well as the fast-twitch fibre formation and muscle tissue patterning in the zebrafish embryo. I present results identifying the previously uncharacterised gene myl1, encoding an alkali-like myosin light chain, which is specifically expressed in fast-twitch muscle progenitors before fibre formation. Furthermore, I introduce data showing that the transcription factor six1 is expressed in Pax7+ muscle progenitor cells, which has been reported to contribute to part of the fast-twitch muscle tissue as well as to a pool of quiescent muscle satellite cells. With support from the presented data, I hypothesise that six1 keeps the Pax7+ muscle progenitor cells in a proliferative state and consequently prevents them from differentiating into muscle fibres. In addition, I demonstrate that the zebrafish fast-twitch muscle fibres can be divided into different subgroups that express unique forms of fast myosin heavy chain genes along the anterior-posterior (head-tail) axis, and that this subspecification depends on a balance between RA and Wnt signalling. Collectively I propose a previously unknown role for Six1 in zebrafish Pax7+ muscle progenitor cell proliferation and differentiation. Furthermore, I present novel data suggesting that distinct regions of the zebrafish body musculature are composed of different fast-twitch fibre types, and that this regionalisation is conserved in adult zebrafish.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

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.

Hindsight (HNT) is a zinc finger transcription factor that is required for morphogenesis of the Drosophila embryo, having roles in germ band retraction (GBR) as well as dorsal closure (DC). HNT expression is also found in sensory organ precursors (SOP) of the developing pupal peripheral nervous system, and muscle progenitor cells, but the role of HNT in neurogenesis and myogenesis during embryogenesis has not been investigated in any depth. Microarray analysis of embryos over-expressing HNT during GBR and DC identified 1290 genes with significant changes in expression. This data set included many potential HNT targets, including genes associated with myogensis, and a disruption of muscle development was observed in embryos over-expressing HNT. It is possible that HNT may function to repress muscle identity genes in muscle founder cells. In addition, HNT over expressing embryos were found to resemble the neurogenic class of mutants. Among the potential target genes, D-Pax2 (shaven, sparkling, CG11049) expression, which is known to be expressed in the developing peripheral nervous system, was confirmed to be up-regulated following HNT over-expression. Interestingly, D-Pax2 and HNT expression were found to co-localize at the onset of their expression at stages 10-12 in embryos, but were not co-localized in later stages of embryogenesis. The up-regulation of D-Pax2 by HNT over-expression was further characterized and was found to be associated with strong ectopic HNT expression. The relevance of HNT to the regulation of D-Pax2 during normal development remains to be determined, but it is possible that endogenous expression of HNT is involved in D-Pax2 repression.

Thesis (Ph. D.)--McMaster University, 2003. / Advisor: Michael Rudnicki. Includes bibliographical references (leaves 187-228). Also available via World Wide Web.