The manipulation of autophagy during early and late reperfusion : the effect on myocardial protection

Smit, Marli (2019-03)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

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.

AFRIKAANSE OPSOMMING: Inleiding: Isgemiese hartsiekte is die hoofoorsaak van wêreldwye sterftes en het ‘n nadelige uitwerking op miokardiale funksie. Die herstel van koronêre bloedvloei, in ‘n poging om die miokardiale selle te red, lei tot herperfusie besering. Die soeke na suksesvolle behandelings opsies teen herperfusie besering is dus van kardinale belang. Die farmakologiese manipulasie van outofagie, ‘n proses wat die georganiseerde afbraak en herwinning van sellulêre komponente behels, is een van die nuutste navorsingsareas wat op kardiale beskerming tydens herperfusie besering fokus. Die belang van outofagie se respons op isgemie en herperfusie, asook die effek van manipulasie daarvan op kardiale beskerming, word steeds gedebatteer. Doelwitte: Ons hipotese is dat induksie van outofagie tydens vroeë herperfusie beskermend, en opregulering van die proses tydens laat herperfusie skadelik is. Die hoofdoel was dus om die beskerming van die hart deur outofagiese induksie tydens vroeë herperfusie en inhibisie tydens laat herperfusie te ondersoek. ‘n Verdere doelwit was om die patroon van outofagiese fluks, wat op verskillende isgemiese en herperfusie periodes volg, te beskryf. Metodes: Geisoleerde harte van manlike Wistar rotte, is geperfuseer volgens die werkhart tegniek. Die kontrole groepe het bestaan uit twee isgemiese (15 en 20 min) en vyf (10, 30, 60, 90 and 120 min) herperfusie intervalle. Tydens die intervensionele eksperimente is 20 min globale isgemie gevolg deur 30 (vroeë) of 120 (laat) min herperfusie. 3 MetielFadenine (3MA) en Rapamycin is toegedien tydens herperfusie om onderskeidelik outofagie te inhibeer en te induseer. Alle eksperimente is herhaal met een uur preFeksperimentele Chloroquine toediening om te onderskei tussen bestendige staat outofagie en outofagiese fluks. Outofagiese proteïenuitdrukking (LC3, Beclin, p62, DRP1, ULK1 en Rab9) aktiwiteit is met behulp van die WesternF klad tegniek geFanaliseer. Hemodinamiese en infarktgrootte bepalings is gebruik om die mate van miokardiale beskerming te bepaal. Resultate: Chloroquine, 3MA en Rapamycin toediening, tydens die intervensionele eksprimente, het nie globale miokardiale funksie voor die aanvang van isgemie beïnvloed nie. WesternFklad: Die kontroles het ‘n toename in outofagiese bestendige staat en fluks tydens herperfusie getoon wat meer uigesproke was met langer isgemiese en herperfusie periodes. 3MA toediening tydens vroeë herperfusie het inhibisie van die tradisionele asook die alternatiewe outofagiese pad, sowel as ‘n toename in apoptose, veroorsaak. Hoë dosering Rapamycin, toegedien tydens laat herperfusie, het ‘n toename in outofagiese flux meegebring, sowel as ‘n inhibisie van die alternatiewe pad tydens beide vroeë en laat herperfusie. Infarkgroottes: Vroeë reperfusie toegediende 3MA het, in vergelyking met al die ander groepe, ‘n beduidende afname in infarkgrootte gedemonstreer. Gevolgtrekkings: Ons eksperimentele model kan suksesvol aangewend word om outofagie te bestudeer, en funksionele outofagie is gedemonstreer tot en met 120 min herperfusie. 3MA toediening tydens vroeë herperfusie is kardiobeskermend wat toegeskryf kan word aan ‘n kombinasie van die inhibisie van die tradisionele en alternatiewe autofagiese paaie, apoptotiese induksie en direkte middelFafhanklike aksies. Laat herperfusie outofagiese induksie het geen nadelige effekte op die hart gehad nie. Outofagiese induksie tydens vroeë reperfusie en inhibisie tydens laat reperfusie was onsuksesvol. Dit word toegeskryf aan beide die beperkte herperfusie periode van die eksperimentele model asook onverwagte probleme ondervind met die farmakologiese manipulasie van outofagie in die betrokke model.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105583
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