Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008. / Cells are equipped with an array of adaptive mechanisms to contest the undesirable effects of
ischemia and the associated hypoxia. Indeed, many studies have suggested that there is an
increase in the PI3K/Akt pathway activation during hypoxia and ischemia. Damaged muscle can
be regenerated by recruiting myogenic satellite cells which undergo differentiation and
ultimately lead to the regeneration of myofibres. The C2C12 murine myogenic cell line is
popular for studying myogenesis in vitro, and has been used in many studies of ischemic
microenvironments. PI3K/Akt pathway activity is increased during C2C12 myogenesis and this
is known to produce an apoptosis resistant phenotype. In this study, we provide evidence that
high basal levels of PI3K activity exist in C2C12 myotubes on day ten post-differentiation.
Ischemia is characterized by depleted oxygen and other vital nutrients, and ischemic cell death is
believed to be associated with an increasingly harsh environment where pH levels decrease and
potassium levels increase. By employing a model that mimics these changes in skeletal muscle
culture, we show that both acute simulated ischemia and acute hypoxia cause decreases in
endogenous levels of the p85 and p110 subunits of PI3K and a consequent reduction in PI3K
activity. Supplementing skeletal muscle cultures with inhibitors of the PI3K pathway provides
evidence that the protective effect of PI3K/Akt is subsequently lost in these conditions. Using
Western blot analysis, a PI3K ELISA assay as well as known inhibitors of the PI3K pathway in
conjunction with the MTT assay we are able to demonstrate that the activation of downstream effectors of PI3K, including Akt, are concurrently decreased during acute simulated ischemia
and acute hypoxia in a manner that is independent of PDK-1 and PTEN and that the decreases in
the PI3K/Akt pathway activity produce a knock-on effect to the downstream signalling of
transcription factors, such as Fox01 and Fox04, in our model. We proceed to provide compelling
evidence that the apoptotic resistance of C2C12s is at least partially lost due to these decreases in
PI3K/Akt pathway activity, by showing increased caspase-3 and PARP cleavage. Then, using
vital staining techniques and a DNA fragmentation assay, we demonstrate increased cell
membrane impairment, cell death and apoptosis after three hours of simulated ischemia and
hypoxia in cultured C2C12 myotubes. In addition to the main findings, we produce evidence of
decreased flux through the mTOR pathway, by showing decreased Akt-dependant
phosphorylation at the level of TSC2 and mTOR during simulated ischemia and hypoxia.
Finally, we present preliminary findings indicating increased levels of HIF1α and REDD-1,
representing a possible oxygen sensing mechanism in our model. Therefore, we show that there
is in fact a rapid decrease in PI3K/Akt activity during severe, acute simulated ischemia and
hypoxia in C2C12 myotubes on day ten post-differentiation, and this causes a concomitant down
regulation in cell survival pathways and increased activity of cell death machinery. Thereafter,
we propose a possible mechanism of action and provide a platform for future studies.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2610 |
| Date | 12 1900 |
| Creators | Thomas, Mark Peter |
| Contributors | Engelbrecht, Anna-Mart, Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | Stellenbosch University |
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