Doctoral Degrees (Medical Physiology)

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Browsing Doctoral Degrees (Medical Physiology) by browse.metadata.advisor "Coetzee, Andre"

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    The manipulation of autophagy during early and late reperfusion : the effect on myocardial protection
    (Stellenbosch : Stellenbosch University, 2019-03) Smit, Marli; Coetzee, Andre; Lochner, Amanda; Strijdom, Hans; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: Introduction: Ischemic heart disease, the leading cause of death worldwide, often has devastating effects on myocardial function. ReFestablishment of coronary flow to salvage myocardial cells results in reperfusion injury. There is an ongoing quest for effective therapeutic interventions against the deleterious effects of this phenomenon. The pharmacological manipulation of autophagy, a process dealing with the organized destruction and recycling of cellular components, is one of the latest focus areas of research on cardioprotection against reperfusion injury. The significance of the autophagic response following ischemia/ reperfusion, as well as the effect of manipulation of this process on cardioprotection is still a matter of debate. Aims: We hypothesised that autophagic induction during early reperfusion is protective while its upregulation during late reperfusion is detrimental. The primary aim was to investigate the protective effect of autophagic induction during early reperfusion and autophagic inhibition during late reperfusion. The secondary aim was to characterise autophagic flux patterns following different ischemic and reperfusion durations. Methods: Isolated hearts from male Wistar rats were perfused in working mode. The control groups consisted of two ischemic (15 and 20 min) and 5 reperfusion (10, 30, 60, 90 and 120 min) intervals. In the interventional experiments 20min of global ischemia was followed by 30 (early) or 120 min (late) of reperfusion, during which 3 MethylFadenine (3MA) and Rapamycin were used to inhibit and induce autophagy respectively. All experiments were repeated with Chloroquine, injection one hour prior to experimentation, to distinguish between steady state and autophagic flux. Western blotting was used to measure autophagic protein levels (LC3, Beclin, p62, DRP1, ULK1 and Rab9). Myocardial protection was measured assessing functional recovery and infarct size. Results: The administration of Chloroquine, 3MA and Rapamycin, in the interventional groups, was without effect on global myocardial function before initiation of ischemia. Western blotting: The control experiments demonstrated an increase in autophagic steady state and flux during reperfusion, being more pronounced following longer ischemic and reperfusion durations. Early reperfusion administered 3MA caused a reduction in conventional and alternative autophagy during early reperfusion, as well as an increase in apoptotic activity. Rapamycin failed to induce autophagy during early reperfusion, but a high dose of Rapamycin resulted in an increase in autophagic flux during late reperfusion. High dose Rapamycin, during early and late reperfusion, additionally resulted in the inhibition of the alternative autophagic pathway. Infarct size: The early reperfusion 3MA group demonstrated a significant decrease in infarct size when compared to all the other groups. Conclusions: Our experimental model can be successfully used to study autophagy, and functional autophagy can be demonstrated up to (at least) 120 min reperfusion. Early reperfusion administered 3MA has cardioprotective properties, this may be attributed to the combination of the inhibition of the conventional and alternative autophagic pathways, apoptotic induction and direct drug effects. Late reperfusion induced autophagy was without effect on cardioprotection. We were unable to convincingly induce autophagy during early reperfusion and inhibit autophagy during late reperfusion, this is mainly attributed to experimental model reperfusion duration restrictions and unexpected difficulties experienced with pharmacological manipulation of autophagy in the model used.