Department of Physiological Sciences
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Browsing Department of Physiological Sciences by browse.metadata.advisor "Durcan, Peter J."
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- ItemThe effects of kirrel1 isoform expression on C2C12 differentiation and fusion in vitro(Stellenbosch : Stellenbosch University, 2023-03) McColl, Rhys Stewart; Myburgh, Kathryn H.; Durcan, Peter J. ; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Adult skeletal muscle myogenesis involves the fusion of muscle progenitor cells into multi-nucleated myofibers, a process crucial for the growth and repair of muscle tissue. Vertebrate myoblast fusion is a relatively poorly understood process that involves a multitude of cell adhesion molecules, actin regulators and fusion proteins. A more comprehensive understanding of myogenesis is essential to better assess muscle myopathies and for the development of improved interventions. The kirrel family of mammalian cell adhesion molecules are highly involved in the production and maintenance of complex tissue structures such as the slit diaphragm in the kidney. The Drosophila paralogs of the kirrel proteins are known to be vital for actin regulation during myoblast fusion with the mechanisms of this regulation being mostly understood. However, these same findings have not been confirmed with regards to mammalian myoblast fusion; an arguably more complex process than that in the fly. It has been demonstrated that kirrel1A and its associated splice variant, kirrel1B, are differentially expressed in regenerating mouse muscle tissue; although, the exact roles of these molecules during this process are not clear. More recently, kirrel3 has been shown to be required for the successful fusion of mouse myoblasts. The aim of this study was to determine the effects of kirrel1A and kirrel1B expression levels on C2C12 differentiation and fusion in vitro. Three genetic strategies were employed to assess kirrel1 activity during C2C12 myogenesis, these being; CRISPR/Cas9 modification, shRNA knockdown and retroviral overexpression. CRISPR/Cas9 was used to disrupt kirrel1 expression by modifying genomic regulatory regions between exons 1 and 2 of the gene. The individual knockdown/inhibition of kirrel1A and kirrel1B mRNA activity was achieved using shRNAs. Overexpression was carried out by wild-type kirrel1A and kirrel1B gene-cloning followed by retroviral transduction. Additionally, a kirrel1A-mCherry mutant was overexpressed in the C2C12s. The differentiation of the various cell lines was assessed via western blotting, PCR analysis and phase-contrast microscopy. The experiments suggest that although the moderate overexpression of kirrel1A or kirrel1B has little effect on myotube production, the gross overexpression of kirrel1 variants leads to a drastic reduction in myogenesis, potentially due to increased steric hindrance at the cell surface. Moreover, our findings demonstrate the requirement for kirrel1A during myotube formation as no tubes were seen in kirrel1A-knockdown myoblasts. This inhibition appeared to be unrelated to the expression of the myogenic regulatory factors. However, it is still unclear whether there is a similar requirement for kirrel1B during fusion. The expression of a mutant form of kirrel1A with an mCherry tag inserted close to an intracellular cleavage site resulted in a complete lack of myotubes; seemingly due to altered early MRF expression. In each case where cell lines produced myotubes, non-reducing western blotting revealed large kirrel1-containing complexes that accumulated as fusion progressed. These complexes were not seen in any of the non-fusing cell lines. From the results it is apparent that kirrel1 is required for healthy myogenesis and that further research is required to fully understand the mechanisms of this regulation.
- ItemInvestigation of myostatin and relevant regulators during muscle regeneration after an acute bout of eccentric exercise(Stellenbosch : Stellenbosch University, 2014-12) Conradie, Johannes David; Myburgh, Kathryn H.; Durcan, Peter J.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: The aim of this study was to investigate the powerful muscle regulator, myostatin, and its regulators in response to an acute bout of plyometric training. The participants were recruited and screened by characterization by means of isometric force production tests, baseline blood creatine kinase levels and VO2 max results. The selected individuals (n=15) were subjected to a baseline muscle biopsy for comparative purposes. The study made use of plyometric jumping, as source of eccentric exercise, to serve as an exercise intervention after which muscle biopsies (4 hours post and 24 hours post) and blood draw (4 hours post, 24 hours post and 48 hours post) samples were taken. Maximal voluntary isometric contractions of the knee extensors were also measured immediately after the exercise protocol and after 1 week recovery. Creatine kinase (CK) analysis on the serum samples was used to conclude muscle damage. The muscle biopsy samples were used for protein quantification (Western blot) and gene expression assessment (semi-quantitative and real-time PCR). The results showed decreased force production immediately after eccentric exercise (p < 0.05), while returning back to baseline values at 1 week post exercise and CK results showed a significant increases at 4 hours (p<0.05), 24 hours (p<0.001) and 48 hours (p<0.01) after exercise. There were no significant differences in myostatin precursor protein (43 kDa), phosphorylated Smad2,3, Smad7 or activin receptor IIb in response to eccentric exercise. However, the follistatin protein was increased at both 4 hours and 24 hours after exercise (p<0.01). RNA analysis of the extracellular matrix (ECM) protein, decorin, revealed the existence of the splice variants A1 and A2 in human skeletal muscle. The RT-PCR analysis (n=4) of these variants showed no significant difference when comparing pre- to post-exercise. The decorin core protein was also investigated by means of antibody probing and results revealed the need for ABC chondroitinase enzyme treatment before immunoblotting of human skeletal muscle samples. The results concerning knee extensor force reduction and circulating creatine kinase showed the effectiveness of plyometric jumping in producing skeletal muscle damage in the lower limbs of unfit individuals, unaccustomed to eccentric exercise. In conclusion, myostatin, and its associated signalling cascade, are not activated in early muscle regeneration, but follistatin is increased during this phase possibly aiding and initiating the muscle repair process. Future studies: Variants of decorin are expressed in human skeletal muscle, increasing the complexity that should be taken into account in studies concerning the regulation of decorin in a human model. Investigation into myostatin protein at different post-translational levels needs more clarification. Published methods and materials used in different laboratories are not consistent and investigators should attempt to standardise protocols in order to compare results between studies more effectively. Of importance, these results show that the myostatin at protein level report different results compared to mRNA analysis and that more investigation into myostatin regulatory factors, with special reference to follistatin and decorin, is needed in future human models.