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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1173 |
| Date | 12 1900 |
| Creators | Loos, Benjamin |
| Contributors | Engelbrecht, Anna-Mart, University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | Unknown |
| Type | Thesis |
| Rights | University of Stellenbosch |
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