Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2009 / ENGLISH ABSTRACT: Three main morphologies of cell death have been described in the diseased myocardium, type I, better known as apoptotic cell death, which is characterized by cell shrinkage and chromatin condensation, type II, or cell death with autophagy, presents a morphology with intracellular accumulation of autophagic vacuoles and type III, better known as necrosis, is characterized by cellular swelling and rapid loss in cellular membrane integrity. However, recent literature strongly argues against rigid classifications in the context of cell death mechanisms but rather suggests to adopt a view of cell death as a dynamic and integrative cellular response. Furthermore, the contribution of autophagy in cell death or cell survival is still poorly understood. Therefore the aims of this study were twofold: (i) to characterize the contribution of each cell death type in context of the severity and duration of an ischaemic insult and (ii) to determine whether manipulation of the autophagic pathway affects the contribution of cell death and translates into protection of the heart. Rodent derived cardiac myoblast cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions. Cells were submitted to protocols of 2, 4 and 8 hrs of simulated ischaemia (SI) under hypoxic conditions in a humidified environment containing 0.1% O2, 5% CO2 and the balance N2, followed by 1 hr of reperfusion respectively. We employed a modified ischaemic buffer containing either 2-deoxy- D-glucose, sodium dithionate or both, with the aim to create an ischaemic insult of mild (mild SI), moderate (moderate SI) and severe (severe SI) character respectively. We evaluated the contribution of each cell death mode using a combination of viability- and ATP assays. Molecular markers for each cell death process such as LC3, PARP and HMGB1 were evaluated using 3-dimensional fluorescence techniques as well as western blot analysis and flow cytometry. Next, autophagy was induced or inhibited prior to the ischaemic insult, using rapamycin and 3MA respectively, and similar parameters were evaluated after 2 hours of mild or moderate SI. Propidium Iodide exclusion and Fluorescence Resonance Energy Transfer (FRET) in combination with mitochondrial inner membrane depolarization were employed to assess the onset of cell death dynamically. Flow cytometry was employed to evaluate the degree of protection. In addition, the ATP levels and reactive oxygen species (ROS) were evaluated. Our results strongly indicate a differential induction of cell death, which is dependent on the severity and duration of the ischaemic insult. Mild SI led to the induction of autophagy and apoptosis, whilst moderate or severe SI induced both apoptotic and necrotic cell death without an indication of autophagy. Only mild SI, but not moderate and severe SI, resulted in an ATP surge. Moreover, our data provide direct evidence that increased autophagy delays the loss of cellular membrane integrity and delays caspase-3 activation as well as mitochondrial depolarization in ischaemic cardiomyocytes. Our results show a profound effect of increased autophagy on the onset of apoptosis as well as necrosis under simulated ischaemic conditions, providing cellular protection. This ATP surge observed during mild SI was abolished with increased autophagy. Furthermore, our results indicate a profound effect of autophagy on ROS generation. Under normoxic conditions, increased autophagy induced a significant decrease in ROS while the inhibition of autophagy significantly increased ROS generation. However, when increasing or decreasing autophagy prior to the ischaemic insult, ROS increased significantly in both scenarios. The results suggest that the severity of ischaemia determines the mode of cell death differentially. An increase in autophagic responsiveness and flux, as induced through rapamycin treatment, provides a selective advantage for tissue against injury, possibly by maintaining intracellular ATP levels through the provision of metabolic substrates. Autophagy is described as an inherent cellular mechanism v which affects the onset of cell death and exhibits protective effects in the ischaemic myocardium when upregulated prior to the ischaemic insult. The protective effect of increased autophagy was mirrored in the isolated perfused rat heart model, reflected by improved functional recovery during ischaemia/reperfusion. / AFRIKAANSE OPSOMMING: Die drie belangrikste morfologiese beskrywings van seldood in die hart sluit die volgende in: tipe I, beter bekend as apoptose wat gekenmerk word deur selkrimping en chromatienkondensering, tipe II, of seldood deur middel van autofagie wat gekenmerk word deur die intrasellulêre versameling van autofagiese vakuole en tipe III, beter bekend as nekrose wat gekenmerk word deur sel swelling en ‘n vinnige verlies aan membraanintegriteit. Onlangse literatuur waarsku egter teen die onbuigsame klassifikasie van seldoodmeganismes en stel voor dat seldood as ‘n dinamiese proses met integrerende sellulêre meganismes beskou moet word. Die bydrae van autofagie in seldoodmeganismes word ook nog nie goed verstaan nie. Die doel van hierdie studie is dus tweevoudig: (i) om die bydrae van elke tipe seldood te bepaal in konteks van die felheid en tydperk van die iskemiese ingryping en (ii) om te bepaal of the manupilering van autofagie ‘n betekenisvolle bydrae lewer in seldoodmeganismes en sodoende tot beskerming van die hart kan lei. Kardiale mioblaste wat van rotweefsel afkomstig is, is in Dulbecco se gemodifiseerde Eagle medium (DMEM), waarby daar 10% fetale kalfserum gevoeg is en wat onderhewig was aan 5% CO2 toestande, onderhou. Selle was onderhewig aan protokolle van 2, 4 en 8 ure gesimuleerde iskemie (SI) onder hipoksiese toestande in ‘n humiditeitsomgewing wat 0.1% O2, 5% CO2 en die balans N2 bevat. Daarna was die selle onderhewig aan 1 uur reperfusie. ‘n Gemodifiseerde iskemiese buffer wat óf 2-deoksie-D-glukose óf natriumdithionaat, of beide bevat, is gebruik om lig, matig en strawwe iskemiese toestande na te boots. Die bydrae van elke tipe seldood is geëvalueer tydens bogenoemde toestande deur gebruik te maak van ‘n kombinasie van sellewensvatbaarheid- en ATP tegnieke. Molekulêre merkers, wat LC3, PARP en HMGB1 insluit, is gebruik om deur middel van 3-dimensionele fluoresensie tegnieke, westelike kladtegnieke en vii vloeisitometrie die verskillende vorme van seldood te ondersoek. Autofagie is ook geïnduseer en geïnhibeer voor die iskemiese ingryping, deur middel van rapamycin en 3MA, respektiewelik om die rol van autofagie tydens seldood te bepaal. Propidium iodite uitluiting en fluoresensie resonansie energie oordrag (FRET) in kombinasie met ‘n merker vir mitochondriale binneste membraan depolarisasie is gebruik om die aanvang van seldood dinamies te ondersoek. Vloeisitometrie is gebruik om die graad van beskerming aan te dui, terwyl intrasellulêre ATP vlakke en reaktiewe suurstof spesies (ROS) ook gemeet is. Ons resultate het getoon dat daar ‘n differensiële indusering van seldood plaasvind wat afhanklik is van die felheid en tydsduur van die iskemiese ingryping. ‘n Ligte graad van iskemie lei tot die indusering van autofagie en apoptose, terwyl matige en strawwe iskemie beide apoptose en nekrose induseer sonder autofagie. Verder het slegs ‘n ligte graad van iskemie ‘n skerp styging in ATP tweeggebring, terwyl dit nie die geval was tydens matige en strawwe iskemie nie. Ons data verskaf ook direkte bewyse dat ‘n toename in autofagie die verlies van sellulêre membraanintegriteit vertraag. Dit het ook ‘n vermindering in caspase-3 aktivering en mitochondriale depolarisasie in iskemiese kardiomiosiete teweegebring. Die data dui aan dat ‘n toename in autofagie ‘n beduidende effek op apoptose en nekrose tydens gesimuleerde iskemiese toestande het om sodoende selbeskerming te verskaf. Die skerp styging in ATP wat tydens die ligte graad van iskemie teweeggebring is, is opgehef met ‘n toename in autofagie. Ons resultate dui ook daarop dat autofagie ‘n beduidende rol in ROS generering speel. Onder normoksiese omstandighede veroorsaak ‘n toename in autofagie ‘n insiggewende afname in ROS generering, terwyl die inhibisie van autofagie ROS generering insiggewend laat toeneem. Wanneer autofagie egter voor die iskemiese ingryping verhoog of verlaag word, vermeerder ROS generering in beide gevalle. Hierdie resultate bewys dat die graad van iskemie ‘n invloed het op die tipe seldood wat geïnduseer word. ‘n Toename in autofagie reaksietyd en vloei, soos viii bewerkstellig deur rapamycin, verskaf ‘n selektiewe voordeel vir weefsel teen beskadiging, heel waarskynlik deur die handhawing van intrasellulêre ATP-vlakke deur die verskaffing van metaboliese substrate. Autofagie word beskryf as ‘n inherente sellulêre meganisme wat seldood beïnvloed en die iskemiese miokardium beskerm wanneer dit opgereguleer word voor die iskemiese ingryping. Hierdie beskermende rol van autofagie wat in die weefselkultuur waargeneem is, is ook in die geïsoleerde geperfuseerde rot hart model waargeneem, waar funksionele herstel verbeter is tydens iskemie/reperfusie.