Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death
worldwide with 3.8 million men and 3.4 million women dying globally each year.
Although existing myocardial reperfusion strategies such as thrombolysis and
percutaneous coronary intervention (PCI), if applied in a timely manner, limit
myocardial infarct size, the mortality and morbidity remains significantly high.
Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial
ischaemia/reperfusion injury through cardioprotective signaling pathways which is
recruited at the time of myocardial reperfusion, thereby improving clinical
outcomes in patients with coronary artery disease.
Ischaemic preconditioning is a phenomenon whereby short intermittent episodes
of coronary occlusion followed by reperfusion protect the myocardium against a
subsequent period of sustained ischaemia. This protection is reflected in the
limitation of infarct size and improved functional recovery of the ischaemic heart
during reperfusion. Despite intensive research efforts, the promise of an effective
cardioprotective strategy using the endogenous protective mechanisms of the
heart which underlies IPC, has not yet been materialized. Although progress has
been made in terms of signaling mechanisms in the preconditioned heart, the
identification of the myocardial reperfusion phase as the critical “window” for
cardioprotection, requires the elucidation of the signal transduction pathways
during the reperfusion phase after IPC.
In view of the above, the aims of the present study were to investigate:
i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC
during early and late reperfusion
ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70
(HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in
the context of IPC during early reperfusion
iii. the involvement of autophagy and apoptosis during early and late
reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of
the heart
v. the mechanism of IPC by means of two-dimensional (2D) proteomics
Methods: The isolated perfused working rat heart model was used with
functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min
global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25
min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when
hearts were snap-frozen for western blotting analysis. Alternatively, hearts were
reperfused for 30 min to record hemodynamic parameters and measure
functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30
min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or
30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts
were reperfused for 30 min to serve as control for the 30 min reperfused IPC
group. Activation of the protein kinases was determined by western blotting
analysis.
For the proteomic study mitochondrial and cytosolic proteins were isolated from
heart tissue and separated in the first dimension by isoelectric focusing, followed
by separation in the second dimension by two dimensional gel electrophoresis.
The PD Quest software programme was used to identify significantly expressed
protein spots. Protein spots of interest were excised and subjected to in-gel
digestion and the resulting peptides were analysed by mass spectrometry.
Proteins were identified by Mascot and the Swiss Prot database.
Results: Western blotting analysis demonstrated that the RISK pathway and p38
MAPK are activated very early in reperfusion, but the activation is not sustained
during the reperfusion period. Autophagy is also upregulated during this early
reperfusion phase; it is attenuated in the middle reperfusion phase and increase
for a second peak of upregulation in the late reperfusion phase. In addition, we
identified CAMKII as a novel marker of functional recovery in IPC after
reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial
and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were
directly related to cardiac metabolism.
Conclusion: Activation of the majority of the protein kinases investigated in the
present study is associated with the hemodynamic parameters of the heart
instead of functional recovery. Results indicated that the variable signaling
patterns could be attributed to differences in heart rate and the effect thereof
(ejection fraction, minimum and maximum rate of contraction), as a result of
sympathetic stimulation due to psychological stress in the animals before
slaughtering. Proteomics results demonstrated that IPC hearts which failed after
ischaemia /reperfusion are metabolically compromised and “worse off” compared
to non-IPC hearts. / AFRIKAANSE OPSOMMING: Inleiding: Koronêre hartsiekte is die vernaamste oorsaak van sterftes wêreldwyd
met 3.8 miljoen mans en 3.4 miljoen vrouens wat jaarliks sterf. Alhoewel
bestaande miokardiale herperfusie strategieë soos trombolise en perkutane
koronêre intervensie (PKI), wanneer betyds toegepas, miokardiale infarktgrootte
beperk, bly mortaliteit en morbiditeit steeds hoog. Isgemiese prekondisionering
(IPK) beskik oor die potensiaal om miokariale isgemie/herperfusie skade te
verminder deur beskermende seinoordragpaaie tydens miokardiale herperfusie te
aktiveer en sodoende die pasiënte wat aan koronêre arterie siekte ly, se
prognose te verbeter.
Isgemiese prekondisionering verwys na die verskynsel waartydens kort episodes
van isgemie opgevolg deur herperfusie, die miokardium teen ‘n daaropvolgende
langdurige isgemiese insident beskerm. Hierdie beskerming word gereflekteer in
die beperking van infarktgrootte en verbeterde funksionele herstel van die
isgemiese hart tydens herperfusie. Ten spyte van intensiewe navorsingspogings
is die presiese meganisme van endogene beskerming tydens IPK nog nie ten
volle ontrafel nie. Die identifisering van die miokardiale herperfusie fase se
kritiese “vensterperiode” van beskerming, noodsaak ‘n volledige analise van die
seinoordragpaaie wat geaktiveer word tydens die herperfusie fase na IPK.
In die lig van bogenoemde, was die doel van die huidige studie om die volgende
te ondersoek:
i. die betrokkenheid van die RISK seinoordragpad en p38 MAP kinase
tydens vroeë en laat herperfusie na IPK
ii. die betrokkenheid van “heat shock protein-27” (HSP-27), “heat shock
protein- 70” (HSP-70), GSK -3β, CAMKII, AMPK en die transkripsie faktor,
CREB, in die konteks van IPK tydens vroeë herperfusie
iii. die betrokkenheid van outofagie en apoptose tydens vroeë en laat
herperfusie na IPK
iv. die korrelasie van die proteïenkinases met die hemodinamiese parameters
van die hart v. die meganisme van IPK deur middel van twee dimensionele proteomika
Metodes: Die geïsoleerde werkende rothart model, met funksionele herstel as
eindpunt, is gebruik. Harte is geprekondisioneer (IPK) met 3x5 min globale
isgemie, afgewissel met 5 min herperfusie. Daarna is harte blootgestel aan 25
min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie,
waartydens harte gevriesklamp is. Alternatiewelik, is harte blootgestel aan 30 min
herperfusie ten einde funksionele herstel te meet en hemodinamiese parameters
te registreer. Nie-geprekondisioneerde (Non-IPK) harte is gestabiliseer vir 30
min, waarna dit onderwerp is aan 25 min volgehoue globale isgemie, gevolg deur
5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is vir westelike
klad analise. Alternatiewelik, is Non-IPK harte onderwerp aan 30 min herperfusie
om te dien as kontrole vir die 30 min IPK groep. Aktivering van die
proteïenkinases is bepaal deur westelike klad analise.
Vir die proteomiese studie, is onderskeidelik mitokondriale en sitosoliese
proteïene geïsoleer en geskei in die eerste dimensie met behulp van isoelektriese
fokusering, gevolg deur skeiding in die tweede dimensie met behulp
van twee dimensionele gel elektroforese. Die PDQuest sagteware program is
gebruik om proteïenkolle te identifiseer wat statisties beduidende verskille toon.
Proteïenkolle van belang is uitgesny en onderwerp aan in-gel tripsinering en die
peptiede wat sodoende verkry is, is deur middel van massa spektrometrie
geanaliseer. Proteïene is geïdentifiseer deur Mascot en die Swiss Prot databasis. Resultate: Westelike klad analise het aangetoon dat die RISK pad en p38 MAPK
geaktiveer is tydens vroeë herperfusie, maar die aktivering word nie volgehou
tydens die hele herperfusie periode nie. Outofagie word gestimuleer tydens die
vroeë herperfusie fase; dit word onderdruk in die middel herperfusie fase en
bereik ‘n tweede piek van stimulering in die laat herperfusie fase. Die
proteomiese analise het onderskeidelik twintig differensieel gereguleerde
mitokondriale proteïene en ses en dertig differensieel gereguleerde sitosoliese
proteïene geïdentifiseer tussen Non-IPK en IPK. Die grootste persentasie van
hierdie proteïene is direk betrokke by miokardiale energie metabolisme. CAMKII
is geidentifiseer as ‘n unieke merker van funksionele herstel in IPK tydens
reperfusie. Gevolgtrekking: Aktivering van die meeste van die proteïenkinases wat
ondersoek is in die huidige studie, is geassosieer met die hemodinamiese
parameters van die hart, in plaas van funksionele herstel. Die resultate het
aangetoon dat die varierende patrone van kinase aktivering toegeskryf kan word
word aan verskille in harttempo en die effek daarvan (ejeksie fraksie, minimum
en maksimum tempo van kontraksie), as gevolg van simpatiese stimulasie
toegeskryf aan sielkundige stres in die diere voor slagting. Proteomiese analise
het getoon dat IPK harte wat faal na isgemie/reperfusie metabolies
gekompromiseer is en “slegter daaraan toe” is, in vergelyking met Non-IPK harte.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85789 |
| Date | 12 1900 |
| Creators | Hattingh, Susanna Maria (Suzel) |
| Contributors | Du Plessis, Stefan, Engelbrecht, Anna-Mart, Salie, Ruduwaan, Stellenbosch University. Faculty of Medical Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xxx, 300 p. : ill. |
| Rights | Stellenbosch University |
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