Aims: The ability of lipopolysaccharide (LPS) pre-treatment to induce cardioprotection following ischaemia/reperfusion (I/R) has been well documented; however, the mechanisms have not been fully elucidated. LPS is a Toll-like receptor 4 (TLR4) ligand. Recent evidence indicates that there is cross-talk between the TLR and phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathways. We hypothesized that activation of PI3K/Akt signalling plays a critical role in LPS-induced cardioprotection. Methods and results: To evaluate this hypothesis, we pre-treated mice with LPS 24 h before the hearts were subjected to ischaemia (45 min) and reperfusion (4 h). We examined activation of the PI3K/Akt/GSK-3β signalling pathway. The effect of PI3K/Akt inhibition on LPS-induced cardioprotection was also evaluated. LPS pre-treatment significantly reduced infarct size (71.25%) compared with the untreated group (9.3 ± 1.58 vs. 32.3 ± 2.92%, P < 0.01). Cardiac myocyte apoptosis and caspase-3 activity in LPS-pre-treated mice were significantly reduced following I/R. LPS pre-treatment significantly increased the levels of phospho-Akt, phospho-GSK-3β, and heat shock protein 27 in the myocardium. Pharmacological inhibition of PI3K by LY294002 or genetic modulation employing kinase-defective Akt transgenic mice abolished the cardioprotection induced by LPS. Conclusion: These results indicate that LPS-induced cardioprotection in I/R injury is mediated through a PI3K/Akt-dependent mechanism.

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.