Thesis (PhD (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / The overall objective of this thesis is to provide additional data to assist clinicians and experimental neurologists alike in the quest for better understanding, more accurately diagnosing and more successfully treating patients suffering from Parkinson’s disease (PD). The general theme of the thesis is the interaction between certain environmental stimuli, including the exposure to adverse events during early central nervous system (CNS) development and the manifestation of elements of neurodegeneration, whether by means of neurochemical changes or expressed as a dysfunctional voluntary motor system. The first chapter provides a general introduction to the research theme of the thesis. This includes, in particular, a discussion on current understanding concerning the etiology and clinical profile of PD, the relative contribution made by genetic factors compared to environmental ones, and current treatment strategies for treating the disease. Mention is also made of the failure of these therapeutic applications for reversing or protecting against the disease, due to the side-effects associated with them. The material covered in chapter 1 provides the basis for the more complete discussion concerning these various aspects, contained in the chapters to follow. The overall aim was also to characterise the effects of commonly used toxin-induced animal models of PD, and the extent of vulnerability that the CNS displays towards them. The destruction of dopaminergic neurons following the administration of 6-OHDA at targeted points along the nigrostriatal tract is used extensively to model PD pathology in rats and is an established animal model of the disease. However, mature or even aged animals are mainly used in these studies, while the effects that the toxin might have on the developing CNS remain unclear. The study reported in chapter 4 aimed to elucidate some of 6-OHDA’s actions on the young adolescent (35 days-old) CNS by comparing the motor and biochemical effects of a unilateral infusion of the toxin into two anatomically distinct basal ganglia loci: The medial forebrain bundle (MFB) and the striatum. Animals were randomly assigned to receive either a direct delivery of 6-OHDA (12μg/4μl) into the MFB or an indirect injection, into the striatum. Although both lesion types were used, the MFB model is considered a more accurate portrayal of end-stage PD, while the striatum-model better reflects the long-term progressive pathology of the disease. The different lesions’ effects on motor function were determined by observing animal’s asymmetrical forelimb use to correct for weigh shifting during the vertical exploration of a cylindrical enclosure. Following the final behavioral assessment, the concentration of dopamine (DA) and DA metabolites remaining in the post-mortem brains were determined using 4 HPLC electrochemistry (HPLC-EC) and the levels compared between the two groups. The HPLC-EC results revealed a compensatory effect for DA production and DA turnover on the lesioned hemisphere side of the toxin-infused animal group. Thus, following 6-OHDA treatment, there appears to be extensive adaptive mechanisms in place within the remaining dopaminergic terminals that may be sufficient for maintaining relatively high extracellular and synaptic concentrations of DA. However, since substantial changes in motor-function were observed, it is suggested that the capacity of the remaining dopaminergic neurons to respond to increased functional demands may be limited. In addition, the behavioral results indicate that the distinct indices relating to different functional deficits depend on the lesioning of anatomically distinct structures along the nigrostrial tract. It has long been known that far fewer women are diagnosed with PD than men are. This seeming protection offered to females against degenerative disease of the CNS may relate to estrogen, although the hormone’s mechanism of action on the dopaminergic system is poorly defined. With an estimated 10-15 million women using oral contraceptives (OCs) in the United States alone, the aim of chapter 2 was to examine the evidence for a possible relationship between PD and the female reproductive hormone estrogen. A review of the current literature available on the topic was performed by consulting Medline, and by performing a search of the case-reports contained within the World Health Organization’s (WHO) International Drug Monitoring database, for possible PD-related symptoms that may arise from estrogen replacement therapy (ERT). The results, whilst conflicting, seem to suggest that estrogen protects women from obtaining the disease, or at least some features of it. Intensive research efforts are called for, with sufficient power to establish the relationship between ERT and the onset and development of parkinsonism. Chapter 3 reports on the results obtained from an experiment that subjected young Sprague-Dawley rats, 35 days of age, to a lower and a higher dose of 6-OHDA delivered to the MFB. Control rats received equivalent saline infusions. At 14 days post-surgery, the rats were evaluated for forelimb akinesia. For the higher dose of 6- OHDA the female rats were less impaired than males in making adjustment steps in response to a weight shift and in the vibrissae-evoked forelimb placing test. In addition, Tyrosine hydroxylase (TH) immunoreactivity was significantly higher for the female rats. Early gender differences in cell survival factors and/or other promoters of neuroplasticity may have contributed to the beneficial outcome seen in the females. For example, nerve growth factor (NGF) was found to be higher in the female rats following administration of the DA neurotoxin. It is unclear whether gonadal steroids are involved, and, if so, whether female hormones are protective or whether male hormones are prodegenerative. Determining the mechanisms for the improved outcome seen in the young female rats may lead to potential treatment strategies against PD. 5 Many studies have shown that early life stress may lead to impaired brain development, and may be a risk factor for developing psychiatric diseases, including clinical depression. However, few studies have investigated the impact that early stress may have on the onset and development of neurodegenerative disorders such as PD. The study reported on in chapter 5 conjointly subjected rat pups to a maternal separation (MS) paradigm that is a well characterised model of adverse early life events, and a unilateral, intrastriatal injection of 6- OHDA. The combined effects of these models on motor deficits and brain protein levels were investigated. Specifically, the animals were assessed for behavioral changes at 28 days postlesion with a battery of tests that are sensitive to the degree of DA loss sustained. The results show that animals that had been subjected to MS display poorer performance in the vibrissae and single-limb akinesia test compared to non-MS control animals (that had also been subjected to the toxin exposure). In addition, there was a significant increase in the loss of TH staining in MS rats compared to non-MS ones. The results from this study therefore suggest that exposure to adverse experiences during the early stages of life may contribute towards making dopaminergic neurons more susceptible to subsequent insults to the CNS occurring during mature stages of life. Therefore, taken together, early exposure to stress may predispose an individual towards the onset and development of neurodegenerative disease, which especially becomes a threat during the later stages of adult life. Moreover, within the framework of these characteristics, the capacity of a widely-used pharmacological agent (statins) was tested for possible future therapeutic application in PD (chapter 7). Although the precise cause of sporadic PD remains an enigma, evidence suggests that it may associate with defective activity of complex I of the mitochondrial electron transport chain. Mitochondrial DNA transmit and express this defect in host cells, resulting in increased oxygen free radical production, depressed antioxidant enzyme activities, and greater susceptibility to apoptotic cell death. Simvastatin is a member of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) group of drugs that are widely used for lowering cholesterol levels in patients who display elevated concentrations of low-density lipoprotein cholesterol. The study aimed to investigate the effects that statin-treatment have on motor-function and at the mitochondrial-protein level, using rotenone, a mitochondrial complex I inhibitor, as a rat-model of PD. Adult male Sprague-Dawley rats were treated either with simvastatin (6mg/day for 14 days) or with a placebo. Two different tests to assess motor function were used: The apomorphine-rotation test, and the vibrissae-elicited forelimb placement test. Following the drug administration protocol, the nigrostriatal tract was unilaterally lesioned with either rotenone (3 μg/4 μl) or, for the controls, were sham-operated by infusing the vehicle (DMSO:PEG) only. Five days later the rats were killed and a highly purified concentration of isolated mitochondria was prepared from the substantia nigra (SN) sections. 2- 6 Dimensional electrophoresis (2-DE) with subsequent identification of the spots using electronspray ionization quadruple time-of-flight mass spectrometrical (ESI-Q-TOF MS) was performed and the results BLAST-searched using bio-informatics tools for naming the identified peptides. The motor test results indicate that while unilateral rotenone causes behavioral asymmetries, treatment with simvastatin improved motor function relative to the rotenoneinduced ones. Mass Spectroscopy identified 23 mitochondrial proteins that differ significantly in protein expression (p < 0.05) following simvastatin treatment. The altered proteins were broadly classified according to their cellular function into 6 categories, with the majority involved in energy metabolism. This study effectively illustrated how neuroproteomics, with its sophisticated techniques and non-biased ability to quantify proteins, provides a methodology with which to study the changes in neurons associated with neurodegeneration. As an emerging tool for establishing disease-associated protein profiles, it also generates a greater understanding as to how these proteins interact and undergo post-translational modifications. Furthermore, due to the advances made in bioInformatics, insight is created concerning their functional characteristics. Chapter 4 summarises the most prominent proteomics techniques and discuss major advances made in the fast-growing field of neuroproteomics in PD. Ultimately, it is hoped that the application of this technology will lead towards a presymptomatic diagnosis of PD, and the identification of risk factors and new therapeutic targets at which pharmacological intervention can be aimed. The final chapter (chapter 8) provides a retrospective look at the academic work that had been performed for the purpose of this thesis, recaps on the main findings, and also highlights certain aspects of the project and provides relevant suggestions for future research. Lastly, the appendix provides a detailed overview of the methods followed for the experiments described in this thesis. It provides not only a comprehensive description of the techniques that had been followed, but provides information concerning the care taken with the animals (i.e. post-surgery) in order to control for the potential influence of experimental variables on the results.

Thesis (PhD (Biomedical Sciences. Medical Physiology.))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Adverse experiences during early life causes alterations in the development of the central nervous system structures that may result in abnormal functioning of the brain. It is well known that, in humans, adverse early-life experiences such as social separation, deprivation, maternal neglect and abuse increase the risk of developing psychiatric disorders, such as depression, later in life. We used maternal separation in the rat as a model for early life stress to firstly determine how different brain systems are dysregulated by this stressful experience and additional chronic or acute stress during adulthood. Rat pups were separated from their mothers on postnatal day 2-14 for 3 hours per day while control rats were normally reared. The behavior, stress response, neurotrophin, apoptotic marker and serotonin levels in the ventral hippocampus, striatum and frontal cortex were measured during adulthood. A different group of maternally separated rats were allowed chronic voluntary exercise and similar measurements were done to determine whether exercise was able to normalize the deficits caused by early life stress. Differentially expressed cytosolic proteins of the ventral hippocampus of maternally separated rats versus normally reared rats were also identified. Protein expression levels of maternally separated rats that received chronic voluntary exercise or escitalopram treatment were subsequently determined to unravel the mechanism of therapeutic action for these two interventions. We found that maternal separation increased the baseline corticosterone response of rats and induced a blunted adrenocorticotropin hormone after acute restraint stress. Baseline neurotrophin levels were significantly decreased in the ventral hippocampus. Maternal separation followed by chronic restraint stress during adulthood resulted in increased depressive-like behavior compared to control rats. Maternal separation alone or followed by acute restraint stress during adulthood induced changes in apoptotic marker expression in the striatum and frontal cortex. In rats subjected to maternal separation and chronic restraint stress during adulthood, we found that chronic voluntary exercise decreased their depressive-like behavior and increased brain derived neurotrophin levels in the striatum. Serotonin levels were not affected by maternal separation, but chronic voluntary exercise increased serotonin in the ventral hippocampus of normally reared rats. Maternal separation induced a number of changes in the expression of cytosolic proteins and these stress-induced changes were identified in proteins relating to cytoskeletal structure, neuroplasticity, oxidative stress, energy metabolism, protein metabolism, and cell signaling. Chronic voluntary exercise was able to restore the expression levels of a number of proteins affected by maternal separation that increased the risk for neuronal death. When comparing the efficacy of exercise to that of escitalopram treatment it was evident that, in contrast to exercise, escitalopram targets a different subset of proteins affected by maternal separation, except for a few involved in energy metabolism pathways and neuroprotection. In this study we have shown that chronic voluntary exercise has therapeutic effects in maternally separated rats, decreasing depressive-like behavior, increasing neurotrophin expression and restoring cytosolic protein expression that were dysregulated by early life stress. / AFRIKAANSE OPSOMMING: Negatiewe stresvolle ervarings gedurende die vroeë stadium van ‘n mens se lewe veroorsaak veranderinge in die ontwikkeling van breinstrukture en het ‘n nadelige uitwerking op die funksionering van die brein. Dit is bekend dat stresvolle ervarings in kinders, byvoorbeeld sosiale afsondering, verwaarlosing en mishandeling, die risiko vir die ontwikkeling van psigiatriese steurings soos depressie gedurende volwassenheid kan verhoog. In hierdie studie gebruik ons moederlike skeiding van neonatale rotte as ‘n model vir vroeë lewensstres om te bepaal hoe dit verskillende sisteme in die brein negatief beinvloed, en dan ook die effek van addisionele kroniese of akute stres gedurende volwassenheid. Die neonatale rotte is weggeneem van hulle moeders af vanaf dag 2 tot 14 vir 3 ure elke dag terwyl kontrole rotte by hulle moeders gebly het. Die gedrag, stres respons, neurotrofiene, apoptotiese merkers en serotonien vlakke is gemeet in die ventrale hippokampus, frontale korteks en striatum gedurende volwassenheid. Rotte wat van hulle moeders geskei is, is dan toegelaat om vir ses weke in wiele te hardloop om te bepaal of kroniese vrywillige oefening die negatiewe effekte wat veroorsaak is deur stres kan ophef. ‘n Bepaling van sitosoliese proteien uitdrukking in die ventrale hippokampus is ook gedoen om die uitwerking van moederlike skeiding op proteienvlak vas te stel. Hierdie protein data is dan vergelyk met die van gestresde rotte wat kroniese oefening of escitalopram behandeling ontvang het om die meganisme van werking van beide behandelings te bepaal. Ons het gevind dat moederlike skeiding die rustende kortikosteroon vlakke van rotte verhoog terwyl dit adrenokortikotropien vlakke na akute stres inhibeer. Moederlike skeiding het ook die neurotrofien vlakke in die ventrale hippokampus verlaag en addisionele kroniese stres gedurende volwassenheid het ‘n verhoging in depressie-agtige gedrag veroorsaak. Moederlike skeiding alleen, sowel as gevolg deur akute stress gedurende volwassenheid het ook veranderinge in die uitdrukking van apoptotiese merkers in die striatum en frontale korteks veroorsaak. Kroniese vrywillige oefening na moederlike skeiding en addisionele stres gedurende volwassenheid kon depressie-agtige gedrag verlaag en neurotrofienvlakke in die striatum verhoog. Serotonien vlakke was nie beinvloed deur moederlike skeiding nie, maar oefening in kontrole rotte het serotonien verhoog in die ventrale hippokampus. Moederlike skeiding het heelwat veranderinge in die uitdrukking van sitosoliese proteiene van die ventrale hippokampus veroorsaak wat ingedeel kan word in die volgende funksionele kategorieë: sitoskelet, neuroplastisiteit, oksidatiewe stres, energiemetabolisme, proteinmetabolisme en seintransduksie. Oefening kon die uitdrukking van verskeie stres-geïnduseerde veranderinge in proteiene weer herstel terwyl dit wou bleik asof escitalopram se meganisme van werking op ‘n ander vlak geskied. Ons bevindinge bewys dat kroniese vrywillige oefening ‘n goeie behandeling is na vroeë lewenstres en dat dit depressiewe gedrag verminder, neurotrofien vlakke verhoog en sitosoliese proteien skeiding alleen, sowel as gevolg deur akute stress gedurende volwassenheid het ook veranderinge in die uitdrukking van apoptotiese merkers in die striatum en frontale korteks veroorsaak. Kroniese vrywillige oefening na moederlike skeiding en addisionele stres gedurende volwassenheid kon depressie-agtige gedrag verlaag en neurotrofienvlakke in die striatum verhoog. Serotonien vlakke was nie beinvloed deur moederlike skeiding nie, maar oefening in kontrole rotte het serotonien verhoog in die ventrale hippokampus. Moederlike skeiding het heelwat veranderinge in die uitdrukking van sitosoliese proteiene van die ventrale hippokampus veroorsaak wat ingedeel kan word in die volgende funksionele kategorieë: sitoskelet, neuroplastisiteit, oksidatiewe stres, energiemetabolisme, proteinmetabolisme en seintransduksie. Oefening kon die uitdrukking van verskeie stres-geïnduseerde veranderinge in proteiene weer herstel terwyl dit wou bleik asof escitalopram se meganisme van werking op ‘n ander vlak geskied. Ons bevindinge bewys dat kroniese vrywillige oefening ‘n goeie behandeling is na vroeë lewenstres en dat dit depressiewe gedrag verminder, neurotrofien vlakke verhoog en sitosoliese proteien vlakke kan herstel.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The Mechanism of -adrenergic preconditioning ( -PC) Ischaemic preconditioning (IPC), a potent endogenous protective intervention against myocardial ischaemia, is induced by exposure of the heart to repetitive short episodes of ischaemia and reperfusion. The protective effects of this phenomenon have been demonstrated to be mediated by release of autocoids such as adenosine, opioids and bradykinin. Release of endogenous catecholamines and activation of the beta-adrenergic receptors (b-AR) have also been shown to be involved in ischaemic preconditioning. However, the exact mechanism whereby activation of the - adrenergic signal transduction pathway leads to cardioprotection, is still unknown. In view of the above, the aims of the present study were to evaluate: (i) the respective roles of the 1-, 2- and 3-AR receptors as well as the contribution of Gi protein and PKA to -adrenergic preconditioning, (ii) the role of the prosurvival kinases, PKB/Akt and ERK 44/p42 MAPKinase in -drenergic preconditioning, (iii) whether b-AR stimulation protect via ischaemia and the formation of adenosine; the respective roles of the A1-, A2-, A3-adenosine receptors as well as the involvement of the PI3-K/PKB/Akt and ERKp44/p42 signal transduction pathways, in the cardioprotective phenomemon of -adrenergic preconditioning and (iv) the contribution of the mitochondrial KATP channels (mKATP), reactive oxygen species and NO to the mechanism of -AR-induced cardioprotection. Methods: Isolated perfused rat hearts were subjected to 35 min regional ischaemia (RI) and reperfusion. Infarct size (IS) was determined using tetrazolium staining (TTC) and data were analyzed with ANOVA. Hearts were preconditioned with 5 min isoproterenol 0.1 μM ( 1/ 2-AR agonist), or formoterol 1 nM ( 2-AR agonist) or BRL 37344 1 μM ( 3-AR agonist) followed by 5 min reperfusion. The roles of the 1-, 2- and 3-ARs as well as NO were explored by using the selective antagonists CGP-20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) and NOS inhibitors L-NAME (50 μM) or LNNA (50 μM) respectively. Involvement of ROS and the mK+ ATP channels was studied by administration of N-acetyl cysteine (NAC, 300 μM) and the mitK+ ATP iv channel blocker 5-HD (100 μM) during the triggering phase. The role of PKA and PI3-K/Akt was investigated by the administration of the blockers Rp-8-CPT-cAMPs (16 μM) and wortmannin (100 nM) respectively, prior to RI or at the onset of reperfusion. Pertussis toxin (PTX), 30 μg kg-1 was administered i.p., 48 h prior to experimentation. The role of adenosine and the adenosine A1, A3, A2A and A2B receptors was studied by using adenosine deaminase and the selective antagonists DPCPX (1 μM), MRS 1191(1 μM), ZM241385 (1 μM) and MRS1754 (1 μM). Activation of PKB/Akt and ERKp44/p42 was determined by Western blot. Results: Infarct sizes of hearts preconditioned with isoproterenol of formoterol were significantly smaller compared to those of non-preconditioned hearts. This was associated with an improvement in postischaemic mechanical performance. However the 3-AR agonist BRL37344 could not reduce infarct size. The 1- and 2-AR blockers CGP-20712A and ICI-118551 completely abolished the isoproterenol-induced reduction in infarct size and improvement in mechanical recovery, while the 3-AR blocker was without effect. Both Rp-8-CPT-cAMPs and wortmannin significantly increased infarct size when administered before 1/ 2-AR preconditioning or at the onset of reperfusion while it reduced mechanical recovery during reperfusion. PTX pretreatment had no significant effect on the reduction in infarct size induced by 1/ 2-AR or 2-AR preconditioning, however it reduced mechanical recovery in the latter. The NOS inhibitors had no effect on the reduction in infarct size induced by 1/ 2-AR preconditioning, but depressed mechanical function during reperfusion. The significant reduction in infarct size by 1/ 2-PC, was associated with activation of ERKp44/p42 and PKB/Akt during the triggering phase, as well as during reperfusion. DPCPX (A1-AdoR antagonist) had no effect on the 1/ 2-PC-induced reduced infarct size or ERK p44/p42 and PKB activation. A2A-AdoR, but not A2b-AdoR, blockade during the trigger phase abolished the reduction in infarct size of 1/ 2-PC. Both antagonists significantly reduced ERK and PKB activation in the trigger phase. In addition, when applied at the onset of reperfusion they significantly reduced ERK p44 / v p42 MAPK and PKB/Akt activation to an even greater extent. MRS-1191 (A3-AdoR antagonist) blocked 1/ 2-PC when applied prior to index ischaemia or when added during early reperfusion, significantly inhibiting both ERK p44 and PKB activation. Cardioprotection of 1/ 2-PC was abolished by inhibition of ROS generation with NAC in the triggering phase as well as at the start of reperfusion. However, the mitoK+ ATP channel blocker 5- HD was without effect. Conclusions: Protection afforded by an acute transient stimulation of the -ARs, depends on the activation of both 1-AR and 2-ARs but not the 3-AR. PKA as well as PI3-K activation prior to sustained ischemia and at the onset of reperfusion were essential for cardioprotection. With functional recovery as endpoint, it appears that NO is involved in 1/ 2-AR preconditioning, while the Gi protein may play a role in 2-AR preconditioning. The production of endogenous adenosine induced by transient b1/b2 stimulation of the isolated rat heart is involved in b−AR preconditioning. Cardioprotection was shown not to be dependent on the A1AdoR while activation of the A3-AdoR occurs during both the triggering and mediation phases. Both the adenosine A2A and, to a lesser extent, the adenosine A2B receptors participate in the triggering phase of b1/b2-PC. Generation of ROS during the triggering and reperfusion phases is involved in eliciting protection, but no role for the mKATP channels could be demonstrated. Finally, activation of the RISK pathway (PKB/Akt and ERKp44/p42) during the triggering phase is a prerequisite for protection. In addition, cardioprotection by b-AR is characterized by activation of the RISK pathway during reperfusion. / AFRIKAANSE OPSOMMING: Iskemiese prekondisionering (IPC) is ‘n kragtige endogene beskerming teen miokardiale iskemie, wat deur blootstelling van die hart aan kort opeenvolgende episodes van iskemie en herperfusie, ontlok word. Hierdie beskerming word medieer deur vrystelling van outakoïede soos adenosine, opioïede en bradikinien. Vrystelling van endogene katekolamiene en aktivering van die betaadrenerge reseptore (b-AR) is bewys om ook by hierdie proses betrokke te wees. Die presiese meganismes waardeur aktivering van die -adrenerge seintransduksiepad tot miokardiale beskerming lei, is nog onbekend. In die lig van bogenoemde, was die doel van die huidige studie om die volgende te evalueer: (i) die onderskeie rolle van die b1-, b2- en b3-AR sowel as die bydrae van die Gi proteïen en PKA in b- adrenerge prekondisionering, (ii) of b-AR stimulasie beskerming ontlok via iskemie en vorming van adenosien, die onderskeie rolle van die A1-, A2-, A3-adenosien reseptore (AdoRs) sowel as die PI3- K/PKB/Akt en ERKp44/p42 seintransduksie paaie, (iv) die mitochondriale KATP (mKATP) kanale, vry suurstof radikale en NO in b−AR prekondisionering. Metodes: Geïsoleerde, geperfuseerde rotharte is aan 35 minute streeksiskemie en herperfusie onderwerp. Infarktgrootte (IS) is deur die tetrazolium (TTC)-kleuringsmetode bepaal. Data is met behulp van ANOVA analiseer. Harte is geprekondisioneer vir 5 min met isoproterenol 0.1 μM ( 1/ 2-AR agonist), of formoterol 1 nM ( 2-AR agonist) of BRL 37344 1 μM ( 3-AR agonist), gevolg deur 5 min herperfusie, voor streeksiskemie. Die belang van die 1-, 2- en 3-ARs sowel as NO is ondersoek, deur onderskeidelik gebruik te maak van selektiewe antagoniste nl CGP- 20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) en NOS inhibitore L-NAME (50μM) of LNNA (50μM). Die rol van die mK+ ATP kanale en ROS is bepaal deur die toediening van die mK+ ATP kanaal blokker 5-HD (100 μM) en die vrye-radikaal opruimer, N-asetiel cysteine (NAC, 300 μM). Die belang van PKA en PI3-K/Akt is bepaal deur toediening van die PKA blokker Rp-8- CPT-cAMPs (16μM) en wortmannin (100nM) respektiewelik. Pertussis toxin (PTX), 30 μg kg-1 is i.p toegedien, 48 uur voor eksperimentasie. vii Die rol van adenosien en die adenosien A1, A2A, A2B en A3 reseptore is bestudeer, deur gebruik te maak van adenosien deaminase en die selektiewe antagoniste DPCPX (1 μM), MRS 1191(1 μM), ZM241385 (1 μM) and MRS1754 (1 μM),repektiewelik. Die middels is deurgaans toegedien tydens die prekondisioneringsprotokol (“snellerfase”) of tydens vroeë herperfusie. Aktivering van PKB/Akt en ERK p44/p42 is deur Western blot analise bepaal. Resultate: Infarktgrootte van harte wat geprekondisioneer is met of isoproterenol ( 1/ 2-PC) of formoterol ( 2-PC), was beduidend kleiner as díe van ongeprekondisioneerde harte. Dit is geassosieer met ‘n toename in postiskemiese meganiese herstel. Die 3-AR agonis BRL37344 ( 3- PC) het egter geen effek op infarktgrootte gehad nie. Die selektiewe 1- en 2-AR blokkers CGP- 20712A en ICI-118551 het die afname in infarktgrootte heeltemal opgehef, asook die verbetering in meganiese herstel tydens herperfusie terwyl die 3-AR blokker geen effek getoon het nie. Beide Rp- 8-CPT-cAMPs en wortmannin het infarktgrootte beduidend vergroot en meganiese herstel beduidend verlaag, wanneer dit net voor 1/ 2-prekondisionering of tydens die begin van herperfusie toegedien is. PTX voorafbehandeling het geen beduidende effek op die vermindering van infarktgrootte (geïnduseer deur 1/ 2-PC of 2-PC) gehad nie. Meganiese herstel is egter verminder in die geval van 2-PC. Die NOS inhibitore het geen effek op die vermindering in infarktgrootte geïnduseer deur b1/b2 gehad nie, maar het ook meganiese herstel onderdruk. Die beduidende afname in infarktgrootte deur b1/b2 prekondisionering is gekenmerk deur aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase. Soortgelyke aktivering van hierdie kinases is ook tydens herperfusie van b-AR geprekondisioneerde harte waargeneem. DPCPX (A1-AdoR antagonis) het geen effek op die infarkt-verminderde effek van 1/ 2- prekondisionering of op ERK p44/p42 en PKB aktivering gehad nie. A2A-AdoR, maar nie A2b – AdoR, blokkade tydens die snellerfase, het die effek van b-AR prekondisionering op infarktgroottee opgehef. Beide antagoniste het die aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase onderdruk. Wanneer toegedien tydens herperfusie, het dit die aktivering van hierdie kinases tot ‘n groter mate onderdruk. MRS-1191 (A3-AdoR antagonis) het infarktgrootte beduidend verhoog en 1/ 2-prekondisionering geblokkeer, beide wanneer dit voor indeks-iskemie toegedien is of tydens vroeë herperfusie, tesame met inhibisie van PKB en ERK p44/p44 aktivering. viii Die kardiobeskerming van 1/ 2-prekondisionering is opgehef deur middel van opruiming van vry suurstof radikale deur NAC in die snellerfase sowel as aan die begin van herperfusie. Die mK+ ATP kanaal blokker 5-HD het geen effek op b-AR prekondisionering gehad nie. Gevolgtrekking: Kardiobeskerming teweeggebring deur ‘n kort periode van stimulasie van die - ARs, is afhanklik van die aktivering van beide 1-AR en 2-ARs, maar nie 3-AR nie. PKA sowel as PI3-K aktivering, net voor volgehoue iskemie en tydens vroeë herperfusie, is aangedui om noodsaaklik vir 1/ 2-AR prekondisionering te wees. Waar funksionele herstel as eindpunt gebruik is, blyk dit dat NO wel betrokke is by 1/ 2-AR prekondisionering, terwyl die Gi protein ‘n rol mag speel in 2-AR prekondisionering. Vorming van endogene adenosien tydens b-adrenerge stimulasie is betrokke by b-AR prekondisionering. Hierdie beskerming is nie van die A1-AdoR afhanklik nie, maar aktivering van die A3-AdoR is nodig tydens beide die sneller en herperfusie fases. Beide die A2A-AdoR, en tot ‘n mindere mate die A2B–AdoR, is ook betrokke by die snellerfase. Vorming van vry suurstof radikale is nodig vir b-AR prekondisionering, nterwyl die mKATP kanale nie betrokke is nie. Ten slotte, aktivering van die RISK seintransduksiepad (ERKp42/p44 en PKB/Akt) tydens die snellerfase is ‘n voorvereiste vir die ontlokking van beskerming. Daarbenewens word b-AR prekondisionering gekarakteriseer deur aktivering van hierdie pad tydens herperfusie. / South African Medical Research Council / University of Stellenbosch

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.

Thesis (MScMedSc)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Obesity is a major risk factor for ischaemic heart disease. Obesity-induced metabolic abnormalities have been associated with increased oxidative stress which may play an important role in the increased susceptibility to myocardial dysfunction and ischaemiareperfusion (I/R) injury seen in obesity. The pineal gland hormone, melatonin, has powerful antioxidant properties. Previous studies have shown that short-term or acute melatonin administration protects the normal healthy heart of lean animals against I/R damage. However, the effects of melatonin on the heart in obesity remain unknown. Moreover, the myocardial signalling mechanisms associated with the cardioprotective effects of melatonin have not been established. Using a rat model of diet induced obesity, we set out to: 1) investigate the effects of chronic melatonin administration on the development of diet-induced systemic alterations including biometric and metabolic parameters and oxidative stress, 2) determine whether chronic melatonin treatment protects the myocardium against ischaemia-reperfusion injury, and 3) determine whether melatonin treatment confers cardioprotection by altering the reperfusion injury salvage kinase (RISK) pathway signalling and the pro-apoptotic p38 MAPK, AMPK and GLUT-4 expression. Male rats weighing 200±20g were randomly allocated to four groups: 1) C, control rats receiving a standard commercial rat chow and drinking water without melatonin; 2) CM, control rats receiving melatonin (4mg/kg/day) in drinking water; 3) D, diet-induced obesity rats, receiving a high calorie diet and drinking water without melatonin; 4) DM, diet-induced obesity rats, receiving melatonin in drinking water. After 16 weeks of treatment and feeding, rats were weighed and blood and myocardial tissue collected to document biochemical and molecular biological changes. Hearts were perfused on the isolated working rat heart perfusion apparatus for the evaluation of myocardial function and infarct size. The Reperfusion Injury Salvage Kinases (RISK) pathway (PKB/Akt (Ser-473), ERK p42/ p44) and p38 MAPK (mitogenactivated protein kinase) were investigated in pre-and post-ischaemic hearts using Western blotting techniques. Post-ischaemic activation of AMPK (5’AMP-activated protein kinase) (Thr- 172) and GLUT-4 (glucose transporter) expression were also investigated. Serum and baseline myocardial glutathione (GSH) content were measured. In addition, serum lipid peroxidation products: thiobarbituric reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxide (LOOH), were also determined. The high-calorie diet caused increases in body weight, visceral adiposity, heart weight, serum insulin, leptin, blood triglycerides, and low HDL-cholesterol levels. Blood glucose levels were similar for both diet fed rats and controls. Myocardial glutathione, serum glutathione, total cholesterol, TBARS, LOOH, CD as well as total cholesterol (TC) levels were not affected by the high calorie diet. Chronic melatonin treatment reduced body weight gain, visceral adiposity, heart weight, blood triglycerides, serum insulin, HOMA index, serum leptin (DM vs D, p<0.01), and increased blood HDL-C in diet treated rats while there was no effect on these parameters in control rats, despite the reduction in body weight, heart weight and visceral adiposity. Melatonin treatment had no effect on myocardial or serum GSH and LOOH in either control or diet animals. It however reduced TBARS and CD in the diet and control groups, respectively. At baseline, chronic melatonin treatment caused a significant increase in phospho-PKB/total PKB ratio and a concomitant reduction in phospho-p38 MAPK/total p38 MAPK ratio of control hearts while there were no such effects on diet-induced-obesity hearts. Infarct size was significantly reduced by melatonin in both diet and control groups (DM: 16.6±2.0%; D: 38.4±2.6% (p < 0.001), and CM: 12.8±1.5%; C: 30.4±1.0%, p<0.001). After coronary artery occlusion and 30 minutes of reperfusion, melatonin increased percentage recovery of aortic output (DM: 28.5±6.5%; D: 6.2±6.2%, p<0.01), cardiac output (DM: 44.4±5.2%; D: 26.6±5.1%, p < 0.01) and total work (DM: 34.5±5.6%; D: 20.4±7.9%, p<0.05) of diet-induced obesity hearts, while having no effect on control hearts. During reperfusion, hearts from melatonin treated rats had increased activation of PKB/Akt (p<0.01), ERK42/44 (p<0.05), and reduced p38 MAPK activation (p<0.05). There was no difference in post-ischaemic activation of AMPK (Thr-172) and GLUT-4 expression in either control or diet fed rats. We successfully demonstrated that chronic melatonin treatment prevented the development of diet-induced metabolic abnormalities and improved ex vivo myocardial function. Melatonin protected the heart against ischaemia-reperfusion injury that was exacerbated in obesity. This was achieved by activation of the RISK pathway. The antioxidant properties of melatonin were involved in these cardioprotective effects. / AFRIKAANSE OPSOMMING: Vetsug of obesiteit is een van die hoof risikofaktore vir iskemiese hartsiekte. Obesiteitgeinduseerde metaboliese abnormaliteite gaan met verhoogde oksidatiewe stres gepaard wat op sy beurt ‘n belangrike rol mag speel in die miokardiale wanfunksie en verhoogde vatbaarheid vir iskemie-herperfusie (I/H) beskadiging, kenmerkend van vetsug. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige anti-oksidant. Vorige studies het getoon dat kort-termyn of akute toediening van melatonien die normale hart van gesonde diere teen I/H beskadiging deur middel van sy anti-oksidant aksies beskerm. Die effek van melatonien op die hart in obesiteit is egter nog onbekend. Hierbenewens is die miokardiale seintransduksie meganismes geassosieer met die beskermende effekte van die hormoon nog nie ontrafel nie. ‘n Model van dieet-geinduseerde obesiteit in rotte is gebruik om die volgende te bepaal: (i) die effek van kroniese melatonientoediening op die ontwikkeling van dieet-geinduseerde sistemiese veranderinge soos biometriese en metaboliese parameters en oksidatiewe stres (ii) die effek van kroniese melatonienbehandeling op die respons van die hart op I/H beskadiging en (iii) die rol van herperfusie beskadiging op die aktivering van PKB/Akt en ERK42/44 (die sg RISK seintransduksiepad), die pro-apoptotiese p38MAPK, AMPK sowel as die uitdrukking van GLUT-4. Manlike Wistar rotte (200±20g) is ewekansig in vier groepe verdeel: (i) C, kontrole rotte wat ‘n standaard rotdieet en drinkwater sonder melatonien ontvang (ii) CM, kontrole rotte wat melatonien (4mg/kg/dag) ontvang (iii) D, dieet-geϊnduseerde vet rotte wat ‘n hoë kalorie dieet en drinkwater sonder melatonien ontvang (iv) DM, dieet-geϊnduseerde vet rotte wat melatonien (4mg/kg/dag) in die drinkwater ontvang. Na 16 weke van behandeling, is die rotte geweeg, bloed en hartweefsel gekollekteer vir biochemiese en molekulêre biologie bepalings. Harte is geperfuseer volgens die werkhartmodel, blootgestel aan iskemie/herperfusie vir evaluering van funksionele herstel en infarktgrootte. Uitdrukking en aktivering van PKB/Akt (Ser-473), ERKp42/p44 en p38MAPK van pre-en postiskemiese hartweefsel is met behulp van Western blot bepaal. Postiskemiese aktivering van AMPK (5’AMP-aktiveerde proteϊen kinase) (Thr-172) en GLUT-4 (glukose transporter) is op soortgelyke wyse bepaal. Serum en basislyn hartweefsel glutatioon (GSH) inhoud asook tiobarbituursuur reaktiewe substans (TBARS), gekonjugeerde diene (CD) en lipiedhidroperoksied (LOOH) konsentrasies is bepaal. Resultate Die hoë kalorie diet het ‘n toename in liggaamsgewig, visserale vet, hartgewig, serum insulien, leptien, plasma trigliseried en lae HDL-cholesterol vlakke teweegebring. Bloed glukosevlakke was egter dieselfde in die vet en kontrole rotte. Miokardiale glutatioon, serum glutatioon, totale cholesterol, TBARS, LOOH, CD is nie deur die dieet beinvloed nie. Chroniese melatonien behandeling het die liggaamsgewig, visserale vet, hartgewig, plasma trigliseried, serum insulien en leptien, HOMA indeks verlaag (DM vs D, p<0.05) en die HDL-cholesterol verhoog in die dieetrotte, terwyl dit geen effek op hierdie parameters in kontrole rotte gehad het nie (uitgesonderd ‘n afname in liggaamsgewig, hartgewig en visserale vet). Melatonien behandeling het geen effek op hart of serum GSH en LOOH in kontrole en vet rotte gehad nie. Dit het egter die TBARS en CD in beide vet en kontrole rotte verlaag. Chroniese melatonien toediening het ‘n beduidende toename in basislyn fosfo PKB//totale PKB ratio en ‘n afname in fosfo p38MAPK/totale p38MAPK ratio teweegebring in harte van kontrole rotte, maar soortgelyke effekte is nie in die harte van die vet rotte waargeneem nie. Infarktgrootte is beduidend deur melatonienbehandeling verlaag in beide dieet en kontrole groepe (DM: 16.6± 5.2%, D: 38.4 ±2.6% (p<0.001); CM: 12.8± 1.5%; C 30.4±1.0 (p<0.001). Na koronere arterie afbinding en 30 min van herperfusie, het melatonien die persentasie herstel van aorta omset (DM: 28.5± 6.5%; D: 6.2± 6.2%, p<0.01), kardiale omset ( DM: 44.4± 5.2%D: 26.6±5.1%, p<0.01) en totale werk (DM: 34.5 5.6%; D 20.4± 7.9%, p<0.05) in die harte van dieetrotte verbeter, terwyl dit sonder effek was in kontrole harte. Tydens herperfusie het harte van melatonienbehandelde rotte verhoogde aktivering van PKB/Akt (p<0.01) en ERKp42/p44 (p<0.05) getoon, terwyl aktivering van p38MAPK verlaag is (p<0.05). Geen verskil in postiskemiese aktivering van AMPK en GLUT-4 uitdrukking is in beide kontrole en dieetrotte waargeneem nie. Ons het daarin geslaag om aan te toon dat chroniese melatonienbehandeling die ontwikkeling van dieet-geϊnduseerde metaboliese abnormaliteite beduidend kan voorkom en ex vivo miokardiale funksie verbeter. Melatonien het ook die hart teen iskemie/herperfusie beskadiging beskerm in beide kontrole en dieetrotte. Bogenoemde veranderinge het met aktivering van PKB/Akt en ERKp42/p44 gepaard gegaan. Die anti-oksidant effekte van melatonien was heelwaarskynlik hierby betrokke.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: HIV/AIDS is a global epidemic that accounts for a large percentage of the mortality in South Africa every year. Since the implementation of anti-retroviral treatment, HIV positive individuals have been living longer, and the cognitive impairment associated with the disease is becoming increasingly apparent. During the initial systemic infection of HIV, the virus migrates through the blood-brain barrier and inflicts axonal injury by causing upregulation of cytokines and neurotoxic proteins. HIV-associated dementia is a neuropsychological classification of cognitive impairment in HIV and a variety of symptoms have been classified as a part of the dementia complex. One of these is apathy, which is thought to be a precursor for dementia in HIV patients. Three groups of individuals have been recruited and scanned using magnetic resonance imaging (MRI) to examine changes in the brain. These are an HIV non-apathetic cohort, an HIV apathetic cohort and a healthy control cohort. Diffusion tensor imaging (DTI) is an MRI technique used to quantitatively assess white matter (WM) integrity using metrics such as fractional anisotropy (FA). Voxel-based analysis, tract-based spatial statistics (TBSS) and tractography are three established DTI analysis methods that have been applied in numerous studies. However, there are certain methodological strengths and limitations associated with each technique and therefore all three of these techniques were used to compare WM differences across groups. The frontal-subcortical pathways are known to be abnormal in apathy, and this has been demonstrated in a number of imaging studies. Most of these studies have examined apathy in the context of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s. However, to our knowledge this is the first DTI study in HIV apathetic patients. With the tractography method, the anterior thalamic radiation and the corpus callosum were reconstructed for each individual to determine whether there were any global changes in these tracts. No significant changes were found. However, a variety of regions in the WM were significantly abnormal in the HIV cohorts when comparing the data at a voxel-based level and using TBSS. This included areas such as the genu and splenium of the corpus callosum, the internal capsule and corona radiata. Changes in frontal WM for the HIV apathy group are an indication of dysfunction in the frontal-striatal circuits, and previous literature has implicated these circuits in the neuropathology of apathy in a variety of central nervous system (CNS) disorders. / AFRIKAANSE OPSOMMING: MIV/VIGS is `n wêreldwye epidemie wat verantwoordelik is vir `n hoë sterftesyfer in Suid- Afrika elke jaar. Sedert die inleiding van anti-retrovirale behandeling, het die MIV-positiewe populasie se lewensduur verleng. Tesame met langer lewensduur, het die kognitiewe verswakking wat geassosieer word met die siekte ook meer prominent na vore gekom. Gedurende die beginstadium van sistemiese infeksie in MIV is daar `n migrasie van die virus deur die bloed-breinskans. MIV kan indirek verantwoordelik wees vir aksonale beskadiging deur verhoging van neurotoksiese proteine en sitokinien te induseer. MIV-geassosieerde demensie is `n neurosielkundige klassifikasie van kognitiewe verswakking in MIV en verskeie simptome is al geïdentifiseer as deel van die demensie kompleks. Een van die simptome is apatie en daar word gespekuleer dat dit `n voorloper is vir demensie in MIV pasiënte. Drie groepe individue was gewerf vir die studie en geskandeer deur magnetiese resonansie beeldvorming (MRB) om sodoende veranderinge in die brein te ondersoek. Die groepe was onderskeidelik `n HIV nie-apatiese kohort, `n HIV apatiese kohort en `n gesonde kontrole kohort. Diffusie tensor beelding (DTB) is `n MRB tegniek wat toegepas word om witstof integriteit te meet deur gebruik te maak van maatstawwe soos fraksionele anisotropie (FA). “Voxel-based analysis”, “tract-based spatial statistics (TBSS)” en “tractography” is drie gevestigde DTB analitiese metodes wat al in talle studies toegepas was. Daar is egter sekere metodologiese voordele en beperkings verbonde aan elke tegniek en daarom is al drie tegnieke gebruik om witstof verskille tussen groepe te vergelyk. Die frontale-subkortikale roetes in die brein is bekend vir abnormaliteite in apatie en dit was ook al gedemonstreer in verskeie studies. Die meeste van die studies het apatie ondersoek in die konteks van neurodegeneratiewe siektes soos Alzheimer se siekte en Parkinson se siekte. Maar sover ons weet is hierdie die eerste DTB studie in MIV pasiënte met apatie. Met die “tractography” metode was die anterior thalamic radiation en corpus callosum herbou vir elke individu. Dit was om te bepaal of daar enige globale veranderinge is in hierdie gebiede, maar geen beduidende veranderinge is gevind nie.`n Verskeidenheid van gebiede in die witstof was beduidend abnormaal in die MIV kohorte wanneer die data vergelyk was met “TBSS” en “voxel-based analysis.” Dit het gebiede ingesluit soos die genu en splenium van die corpus callosum, die internal capsule en die corona radiata. Veranderinge in die frontale witstof vir die MIVapatie groep is `n aanduiding van disfunksie in die frontale-striatale bane. Vorige literatuur impliseer dat hierdie bane betrokke is in die neuro-patologie van apatie in verskeie sentrale senuweestelsel (SS) steurings.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Primary culture of isolated adult rat cardiomyocytes (ARCMs) is an important model for cardiovascular research, but successful maintenance of these cells in culture for their use in experiments remains challenging (Xu et al, 2009; Louch et al, 2011). Most studies are done on acutely isolated cardiomyocytes immediately after isolation, which is due to low survival of these cells in culture. Obstacles in culture are due to the type of medium and attachment factors (tissue culture adhesives) used to culture and grow these cells. Although we previously identified an optimum medium and adhesive for culture, an adhesive that permits cells to remain attached to the culture surface until after an ischemia/reperfusion insult was elusive. Aims: We therefore aimed to identify the best attachment factor and concentration that will allow adult rat cardiomyocytes to remain attached to the culture surfaces after ischemia/reperfusion experiments. Methods: Cardiomyocytes were isolated from adult Wistar rat hearts and cultured overnight on different concentrations (25 -200 μg/ml) of collagen 1, collagen 4, extracellular matrix (ECM), laminin/entactin (L/E) and laminin. Following overnight cultures, experiments were done in PBS and in PBS versus MMXCB to compare ARCM attachment and viability. Cardiomyocytes cultured on ECM, L/E and L (25−200μg/ml) were subjected to 1 hour of simulated ischemia using MMXCB that contained 3mM SDT and 10mM 2DG, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view of 1.17μm/mm2 were captured using fluorescence microscopy. The cells were analysed according to morphology and fluorescence intensity. Results: Total and rod-shaped ARCMs attachment was improved when MMXCB was used as an experimental buffer instead of PBS. Regardless of the buffer used, morphological viability was poor on substrates of Col 1 and Col 4. In contrast to collagens, ARCMs attached efficiently and morphological viability was high on substrates of ECM, L/E and L in MMXCB, but this was greatly reduced in PBS. Mitochondrial viability was high in MMXCB compared to PBS on Col 1 and Col 4 at 75−175μg/ml and on ECM, L/E and L at all concentrations, except at 50 and 150μg/ml ECM, 175μg/ml L/E and 25μg/ml L. When cardiomyocytes cultured on ECM, L/E and L were subjected to simulated ischemia, total ARCMs, rod-shaped and R/G fluorescence (mitochondrial viability) was reduced at all concentrations compared to the control group. Hypercontracted cells were higher in the ischemic treated cells compared to the controls on ECM at 75−150μg/ml and 200μg/ml, L/E at 50,100μg/ml and 175μg/ml and on L at 125μg/ml. Total numbers of ARCMs attached on ECM, L/E and L in the ischemic group consisted of similar numbers of non-viable hypercontracted and viable rod-shaped cells. Conclusion: Cardiomyocytes should be cultured on ECM or L/E or L at concentrations from 25−200μg/ml in MMXCB. PBS is harmful to cultured ARCMs and should thus not be used as an experimental buffer. Ischemia/reperfusion can be simulated on ARCMs cultured on ECM, L/E or L from 25−200μg/ml, provided that a modified culture buffer is used as experimental buffer. / AFRIKAANSE OPSOMMING: Inleiding: Primêre selkulture van geïsoleerde volwasse rot kardiomiosiete (VRKMe) is ‘n belangrike model vir kardiovaskulêre navorsing, maar om hierdie selle suksesvol in kultuur te onderhou is ‘n groot uitdaging (Xu et al, 2009; Louch et al, 2011). Die meeste navorsingstudies maak gebruik van akuut geïsoleerde kardiomiosiete onmiddelik na isolasie omdat oorlewing van hierdie selle in kultuur baie laag is. Die struikelblokke in kultuur is as gevolg van die tipe medium en weefselkultuurgom wat gebruik word. Ons het voorheen 'n optimale medium en weefselkultuurgom geïdentifiseer vir VRKM kultuur oorlewing, maar die weefselkultuurgom was nie effektief genoeg om die selle aan die kultuuroppervlak te laat bly vaskleef, tot na die einde van 'n isgemie/herperfusie eksperiment nie. Doel: Die doel was dus om die beste weefselkultuurgom en konsentrasie te identifiseer, wat sal toelaat dat VRKMe verbonde bly aan die kultuuroppervlaktes tot na die einde van isgemie/herperfusie eksperimente. Metodes: Kardiomiosiete was geïsoleer vanaf volwasse Wistar rotharte en oornag in kultuur op verskillende konsentrasies (25 -200 μg/ml) van kollageen 1, kollageen 4, ekstrasellulêre matriks (ESM), laminin/entactin (L/E) en laminin onderhou. Die volgende dag was die VRKMe vir eksperimentasie in PBS en in PBS teenoor MMXCB gebruik, om selbehoud en oorlewing te vergelyk. Kardiomiosiete op ESM, L/E en L (25−200μg/ml) was aan 1 uur van gesimuleerde isgemie blootgestel, in MMXCB wat 3mM SDT en 10mM 2DG bevat het, gevolg deur 15 minute herperfusie. Sel oorlewing was bepaal deur selle te kleur met JC-1 en daarna was fluoressensiebeelde van die selle in ‘n veldgebied van 1.17μm/mm2 geneem. Die selle was volgens selmorfologie en fluoressensie intensiteit ontleed. Resultate: Met die gebruik van MMXCB as eksperimentele buffer in plaas van PBS, het die aantal totale en staafvormige VRKMe verbinding verbeter. Morfologiese onderhoud was sleg op kollageen 1 en 4, ongeag van watter buffer gebruik was. In kontras met die kollagene was die VRKM verbinding en morfologiese onderhoud op ESM, L/E en L in MMXCB effektief verbeter, maar in PBS aansienlik verminder. Mitochondriale lewensvatbaarheid in MMXCB teenoor PBS op kollageen 1 en 4 by 75−175μg/ml, sowel as op ECM, L/E en L by alle konsentrasies, was hoog, behalwe by 50 en 150μg/ml ESM, 175μg/ml L/E en 25μg/ml L. Isgemie blootstelling van kardiomiosiete gekultuur op alle konsentrasies van ESM, L/E en L, het ‘n afname in die totale, staafvormige en R/G fluoressensie (mitochondriale lewensvatbaarheid) teweeggebring. Meer hiperkontrakteerde kardiomiosiete was in die isgemie behandelde groepe as in die kontrole groepe teenwoordig, spesifiek op ESM by 75−150μg/ml en 200μg/ml, op L/E by 50,100μg/ml en 175μg/ml asook op L by 125μg/ml. In die isgemie groepe het die totale aantal VRKMe op ESM, L/E en L meestal uit ‘n gelyke hoeveelheid hiperkontrakteerde en staafvormige selle bestaan. Gevolgtrekking: Kardiomiosiete moet op ESM of L/E of L by konsentrasises van 25−200μg/ml in MMXCB gekultuur word. PBS is nadelig vir VRKMe in kultuur en moet dus nie gebruik word as eksperimentele buffer nie. Isgemie/herperfusie eksperimente kan gesimuleer word op VRKMe wat op 25−200μg/ml ESM, L/E of L gekultuur is, mits ‘n gemodifiseerde kultuur buffer gebruik word as eksperimentele buffer.

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: The cardioprotective actions of the hormone melatonin against myocardial ischaemiareperfusion injury (IRI) are well-established. It has recently been shown to prevent the harmful effects of hyperphagia-induced obesity on the susceptibility of the heart to IRI as well as many of the harmful effects of obesity and insulin resistance. However, the exact mechanism whereby it exerts its beneficial action is still unknown. The aims of this study were to determine the effects of relatively short-term melatonin treatment in a rat model of diet-induced obesity on: (i) biometric and metabolic parameters, lipid peroxidation, myocardial IRI and intracellular signalling (ii) mitochondrial oxidative phosphorylation function (iii) cardiomyocyte glucose uptake and intracellular signalling. In addition, the effects of acute melatonin treatment of cardiac microvascular endothelial cells (CMEC) were determined on cell viability, nitric oxide production (NO), TNF- -induced dysfunction and intracellular signalling. Material and Methods: Male Wistar rats were randomly allocated to two groups for 20 weeks feeding with either standard rat chow or a high calorie diet. Each group was subdivided into 3 groups receiving either water throughout or melatonin (4mg/kg/day, in the drinking water) for the last 6 or 3 weeks of the experimental programme. Hearts, perfused in the working mode, were subjected to ischaemia/reperfusion and infarct size determined. Mitochondria and cardiomyocytes were isolated according to standard techniques and oxidative function and glucose uptake respectively determined. CMEC NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide/Annexin V respectively. Intracellular signalling was evaluated using Western blot and appropriate antibodies. Results: The high-calorie diet caused significant increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin, triglycerides, HOMA-IR index and a concomitant reduction in serum adiponectin levels as well as larger myocardial infarct sizes after exposure to IRI compared to the control, indicating increased susceptibility to damage. Three as well as six weeks of melatonin administration to obese and insulin resistant rats reduced serum insulin levels and the HOMA-IR index. Myocardial infarct size was reduced in both control and diet groups. These effects were associated with increased activation of baseline myocardial STAT- 3 and the RISK pathway during reperfusion. The diet had no effect on the oxidative phosphorylation capacity of mitochondria, isolated from non-perfused hearts (baseline), but melatonin administration for 6 weeks induced a reduction in state 3 respiration rate; mitochondria isolated from diet hearts subjected to global ischaemia, exhibited an attenuated oxidative phosphorylation process which was improved by melatonin treatment. Melatonin in vitro enhanced cardiomycyte insulin stimulated glucose uptake of normal young rats but not of insulin resistant rats. In vivo melatonin treatment for 6 weeks increased basal (in diet group) and insulin stimulated glucose uptake in both control and diet groups. Melatonin (1nM) in vitro caused a significant reduction in necrosis and apoptosis of cultured CMEC, associated with a decrease in nitric oxide availability and eNOS activation and a concomitant increase in PKB/Akt, p38MAPK and AMPK activation. The harmful effects of TNF- treatment on signalling in CMEC could be prevented by co-treatment with melatonin. Conclusions: The results suggest that short-term melatonin treatment was able to significantly attenuate the diet-induced increased myocardial susceptibility to ischaemia/reperfusion damage. It may also improve cardiac glucose homeostasis and mitochondrial oxidative phosphorylation in an insulin resistant state. Melatonin in vitro protects CMEC against apoptosis and necrosis and reduces nitric oxide availability. These beneficial effects of melatonin may ultimately be due to its antioxidant capacity or receptor-mediated actions, but this remains to be established. / AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van die hormoon, melatonien, om die hart teen iskemie/ herperfusiebesering (IHB) te beskerm, is welbekend. Onlangs is ook getoon dat melatonien IHB en verskeie van die nadelige effekte van vetsug en insulienweerstandigheid in hiperfagiegeïnduseerde vetsug kan voorkom. Die meganisme(s) betrokke by hierdie voordelige prosesse is egter grootliks onbekend. Die doel van hierdie studie was om die gevolge van korttermyn melatonienbehandeling in ‘n model van hiperfagiegeïnduseerde vetsug te ondersoek op (i) biometriese en metaboliese parameters, lipiedperoksidasie, miokardiale IHB en intrasellulêre seintransduksie, (ii) mitochondriale oksidatiewe fosforilasie, (iii) glukoseopname en intrasellulêre seintransduksie in kardiomiosiete en aanvullend, (iv) die invloed van akute melatonienbehandeling van kardiale mikrovaskulêre endoteelselle op sellulêre oorlewing, stikstofoksiedproduksie, TNF- - geïnduseerde disfunksie en seintransduksie. Metodiek: Manlike Wistarrotte is ewekansig in twee groep verdeel en vir 20 weke met standaard-rotkos of ‘n hoëkaloriedieet gevoer. Elke groep is in 3 subgroepe verdeel, wat deurgaans water of melatonien (4mg/kg/dag in die drinkwater) vir 3 of 6 weke voor die beëindiging van die eksperiment ontvang het. Harte is geperfuseer volgens die werkharttegniek, blootgestel aan iskemie/herperfusie en die infarktgrootte bepaal. Mitochondria en kardiomiosiete is volgens standaardtegnieke geïsoleer vir bepaling van oksidatiewe funksie en glukoseopname respektiewelik. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied/Annexin V onderskeidelik. Intrasellulêre seintransduksie is evalueer met behulp van die Western kladtegniek en geskikte antiliggame. Resultate: Die hoëkaloriedieet het ‘n beduidende toename in liggaamsgewig, visserale vet, vastende bloedglukose, seruminsulienvlakke, trigliseriede, HOMA-IR-indeks en ‘n gepaardgaande verlaging in serumadiponektienvlakke tot gevolg gehad, sowel as groter miokardiale infarkte na iskemie/herperfusie. Laasgenoemde dui op ‘n groter vatbaarheid vir iskemiese beskadiging in harte van vetsugtige diere. Drie sowel as ses weke van melatonienbehandeling het die seruminsulienvlakke en HOMAindeks in vetsugtige diere beduidend verlaag, vergeleke met die kontroles. Miokardiale infarktgroottes was verminder in beide kontrole- en vetsuggroepe. Hierdie effekte het met ‘n verhoogde aktivering van basislyn STAT-3 en PKB/Akt en ERKp44/p42 tydens herperfusie gepaard gegaan. Die dieet het geen invloed op die oksidatiewe fosforilasiekapasiteit van mitochondria, geïsoleer uit harte van ongeperfuseerde harte, gehad nie (basislyn), maar melatonienbehandeling vir 6 weke het Staat 3 respirasie verlaag. Mitochondria, geïsoleer uit harte van vetsugtige rotte wat aan globale iskemie onderwerp was, het ‘n onderdrukte oksidatiewe fosforilasieproses gehad, wat egter deur melatonienbehandeling verbeter is. Melatonien in vitro het insuliengestimuleerde glukoseopname deur kardiomiosiete van jong, maar nie vetsugtige rotte nie, verhoog. In vivo melatonientoediening vir 6 weke het egter basale (in die dieetgroep) en insuliengestimuleerde glukoseopname in beide kontrole- en vetsuggroepe verhoog. Toediening van melatonien in vitro aan mikrovaskulêre endoteelselkulture het ‘n beduidende afname in nekrose, apoptose, stikstofoksied- beskikbaarheid en eNOS aktivering teweeggebring, tesame met ‘n verhoogde aktivering van PKB/Akt, p38MAPK en AMPK. Die nadelige effekte van TNF- toediening op seintransduksie in die mikrovaskulêre endoteelselle is deur melatonien voorkom. Gevogtrekkings: Die resultate toon dat melatonien ‘n merkwaardige beskermende effek op die toename in vatbaarheid vir iskemiese beskadiging in vetsugtige rotte gehad het. Dit mag ook miokardiale glukose-homeostase en mitochondriale oksidatiewe funksie in insulienweerstandigheid verbeter. Melatonien in vitro beskerm mikrovaskulêre endoteelselle teen nekrose asook apoptose en verminder die beskikbaarheid van stikstofoksied. Hierdie voordelige effekte van melatonien mag aan sy anti-oksidantvermoëns of stimulasie van die melatonienreseptor toegeskryf word, maar bewyse daarvoor ontbreek nog. / Division of Medical Physiology (Stellenbosch University), / National Research Foundation / Harry Crossley Foundation

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Ischaemic heart disease is a major contributor to global morbidity and mortality rates. Manoeuvres such as ischaemic preconditioning confer cardioprotection against ischaemia/reperfusion (I/R) injury by activating several intracellular signalling pathways. These pathways have been defined solely in terms of the kinases involved, despite the realization in recent years that protein phosphatase activity also contributes significantly to the attributes of the propagated signal. Protein phosphatase 2A (PP2A) is a heteromultimeric enzyme involved in an array of phosphatase reactions. We hypothesized that PP2A is an important participant in the myocardial response to I/R by regulating intracellular signalling. This project aimed to (i) characterize PP2A during myocardial I/R; (ii) determine the importance of its contribution to the cellular response to I/R; and (iii) investigate its role in the signalling pathways mediated by PKB/Akt, GSK-3β, ERK p42/p44 and p38 MAPK. Two models were used to characterize PP2A during I/R: (i) H9c2 cells exposed to simulated ischaemia (SI) buffer in conjunction with hypoxia (0.5% O2) for a maximum of 2 hours, followed by reoxygenation in standard growth medium for up to 30 minutes; and (ii) isolated working rat hearts exposed to a maximum of 20 minutes global ischaemia and 10 minutes reperfusion. In both models samples were collected at several time points during I/R for Western blotting analysis. PP2A-C (the catalytic subunit) accumulated in the nucleus during early ischaemia, but later redistributed to the cytosol. At the end of ischaemia there was an elevation of PP2A-C relative to PP2A-A in the unfractionated whole cell preparation concomitant with an increase in the inhibitory phosphorylation of PP2A-C. The impact of PP2A activity was evaluated by either inhibiting PP2A using okadaic acid (OA, 10 nM) or activating it by administering FTY720 (1 μM) in an isolated working rat heart model exposed to either 35 minutes of regional ischaemia (RI) with infarct size (IFS) as primary end-point, or 20 minutes global ischaemia (GI) with functional recovery as end-point. The results showed that the pre-ischaemic administration of OA or FTY720 reduced or exacerbated IFS respectively, indicating that PP2A activation during I/R favours cell death. OA and FTY720 were also employed to assess the contribution of PP2A to intracellular signalling in an isolated working rat heart exposed to I/R. Samples were collected at several timepoints and analyzed using Western Blotting. Pre-ischaemic administration of OA enhanced the phosphorylation of PKB/Akt, ERK p42/p44 and GSK-3β at the onset of reperfusion, while FTY720 given before ischaemia reduced the phosphorylation of GSK-3β, p38 MAPK and PKB/Akt at the end of ischaemia and onset of reperfusion. In summary, PP2A is part of an early nuclear-based response to ischaemia, while long-term ischaemia induces an increase in PP2A-C. A portion of this PP2A-C is stored in an inactive form, while an active portion acts as a regulator of the pro-survival signalling components PKB/Akt, GSK- 3β and ERK p42/p44 at the end of ischaemia and the onset of reperfusion. PP2A is therefore an important component of the myocardial response to I/R by regulating pro-survival signalling. / AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die belangrikste komponente wat bydra tot globale morbiditeit en mortaliteit. Ingrepe soos iskemiese prekondisionering aktiveer veelvoudige intrasellulêre seintransduksiepaaie om kardiobeskerming teen iskemie/herperfusie (I/H)-besering te ontlok. Die kinases betrokke in hierdie seintransduksiepaaie is reeds deeglik nagevors, terwyl die potensiële belang van die proteïenfosfatases in seintransduksie tot onlangs misken is. Ons hipotese was dat Proteïenfosfatase 2A (PP2A), wat in ‘n wye verskeidenheid fosfatase reaksies betrokke is, ‘n belangrike rolspeler in die miokardiale reaksie op I/H-besering is, deur deelname aan die regulering van intrasellulêre seintransduksie. Hierdie projek het ten doel gehad om (i) PP2A te karakteriseer tydens miokardiale I/H; (ii) die belang van PP2A in die sellulêre reaksie op I/H-besering te bepaal; en (iii) PP2A se rol in die seintransduksiepaaie, gemedieer deur PKB/Akt, GSK-3β, ERK p42/p44 en p38 MAPK, te evalueer. Twee modelle is aangewend om PP2A tydens I/H te karakteriseer: (i) H9c2-selle blootgestel aan ‘n simuleerde iskemiebuffer tesame met hipoksie (0.5% O2) vir ‘n maksimum van 2 uur gevolg deur heroksiginasie in standaardgroeimedium vir verskillende tydsperiodes tot ‘n maksimum van 30 minute; en (ii) geïsoleerde, werkende rotharte blootgestel aan ‘n maksimum van 20 minute globale iskemie en 10 minute herperfusie. In beide modelle is monsters op verskillende tye versamel vir Western-kladanalise. Tydens vroeë iskemie het PP2A-C in die kern toegeneem, waarna dit met verloop van tyd na die sitosol herversprei het. Teen die einde van iskemie was daar ‘n toename in die vlakke van PP2A-C relatief tot PP2A-A in ongefraksioneerde weefselhomogenate, tesame met ‘n toename in die inhibitoriese fosforilering van PP2A-C. Die belang van PP2A-aktiwiteit is ondersoek deur die effek te bepaal van die inhibisie of aktivering daarvan op infarktgrootte (IFS) en funksionele herstel in ‘n geïsoleerde werkende rothartmodel, blootgestel aan onderskeidelik 35 minute streeksiskemie (RI) of 20 minute globale iskemie. Preiskemiese toediening van die PP2A-inhibitor okadaïensuur (OA, 10 nM), of aktiveerder FTY720 (1 μm) het infarktgrootte respektiewelik beperk of vergroot. PP2A-aktivering tydens I/H is dus nadelig. OA en FTY720 is ook aangewend om die bydrae van PP2A tot I/H-verwante, intrasellulêre seintransduksie in die geïsoleerde, werkende rothart te bepaal. Monsters is op verskeie tydintervalle versamel en ontleed deur gebruik te maak van die Western-kladtegniek. Preiskemiese toediening van OA het die fosforilering van PKB/Akt, ERK p42/p44 en GSK-3β by die aanvang van herperfusie bevoordeel, terwyl pre-iskemiese toediening van FTY720, die fosforilering van GSK-3β, p38 MAPK en PKB/Akt aan die einde van iskemie en die begin van herperfusie verminder het. Ter opsomming: PP2A is deel van ‘n vroeë gelokaliseerde kerngebaseerde reaksie op iskemie, terwyl langdurige iskemie ‘n toename in PP2A-C relatief tot PP2A-A induseer. ‘n Deel van hierdie PP2A-C is onaktief, terwyl die res funksioneer in die regulering van die seintransduksiekomponente PKB/Akt, GSK-3β en ERK p42/p44 wat oorlewing fasiliteer met die aanvang van herperfusie. PP2A is dus ‘n belangrike komponent in die miokardiale reaksie op I/H deurdat dit tot die beheer van seintransduksiepaaie bydra.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / Background and purpose. To limit ischaemic injury, rapid restoration of coronary blood flow is required, which will in turn reduce infarct size. However, reperfusion itself causes myocyte death – a phenomenon termed lethal reperfusion-induced injury, which limits protection of the ischaemic myocardium. Thus the reperfusion of irreversibly damaged myocytes may accelerate the process of cell necrosis. Additive protection of the ischaemic myocardium in the form of adjunct therapy remains a topic of intensive research. Levosimendan, a calcium sensitizing agent with positive inotropic effects has in several studies been found to alleviate the damaging effects of reperfusion injury. Levosimendan has been shown to be a KATP channel opener. These channels have been implicated to play an important role in ischaemic preconditioning (IPC). With this knowledge, the aim of this study was to determine whether levosimendan and IPC have certain cardioprotective mechanisms in common and whether protection with pharmacological preconditioning could be elicited with levosimendan. In this study, we investigated whether: 1) the isolated guinea pig heart could be protected by ischaemic preconditioning (IPC) and postconditioning (IPostC), 2) the heart could be pharmacologically pre- and postconditioned, using levosimendan (LPC & LPostC), 3) a combination of IPC & LPC had an additive protective effect on the heart, 4) the KATP (both mitochondrial and sarcolemmal) channels are involved in this protection and 5) the pro-survival kinases of the RISK (reperfusion injury salvage kinase) pathway are involved. Experimental approach. Isolated perfused guinea pig hearts were subjected to three different IPC protocols (1x5, 2x5 and 3x5 minutes of ischaemia) or levosimendan (0.1μM) preconditioning, before coronary artery occlusion (CAO – 40min@36.5ºC), followed by 30 minutes of reperfusion. Hearts were also subjected to a combination of IPC & LPC, to establish whether they had additive protective effects. In addition, hearts were pre-treated with levosimendan directly before induction of sustained ischaemia (without washout of the drug – levosimendan pre-treatment (LPT)) for 10min. With the postconditioning protocol, iii the hearts were subjected to 3x30second cycles of ischaemia/reperfusion or levosimendan/vehicle. In a separate series of experiments, hearts were treated with KATP channel blockers (for both sarcolemmal & mitochondrial), before LPC, LPT and LPostC. The endpoints that were measured were: cardiac reperfusion function, myocardial infarct size and RISK pathway expression and phosphorylation (PKB/Akt and extracellular signal-regulated kinase – ERK42/44). Results. IPC, IPostC, LPC & LPostC decreased myocardial infarct size significantly compared with their controls (21.9±2.2%, 21.4±2.2%, 20.6±3.1% and 20.6±1.8% respectively vs. 46.4±1.8% for controls, p<0.05). The combination of IPC & LPC had no additive protective effect. Pre-treating the hearts with levosimendan (without washout), before index ischaemia, proved to be the most effective method of cardioprotection (infarct size: 5.8±0.9% vs. 46.4±1.8% for controls, p<0.001). With LPT a significant increase (p < 0.05 vs. control) in phosphorylation of ER42/44 was also observed. An increase in the activity of one of the RISK pathway kinases, ERK42/44 seems to be one of the reasons for LPT’s efficacy. Treating the hearts with KATP channel blockers before subjecting them to LPC, LPT & LPostC abolished the protective effects induced by levosimendan, suggesting a role for the sarcolemmal and mitochondrial KATP channels in levosimendan-induced cardioprotection. Conclusions and implications. 1) Isolated guinea pig hearts could be pre- and postconditioned within the setting of ischaemia, 2) Hearts could be pharmacologically pre- and postconditioned with levosimendan, 3) levosimendan pre-treatment is the most effective way to reduce infarct size, possibly acting by increasing the phosphorylation of ERK42/44, 4) Myocardial protection was not increased by combining IPC & LPC (suggesting similar mechanisms of protection), 5) LPC, LPT and LPostC were abolished by both sarcolemmal and mitochondrial KATP channel blockers. .LPC and especially LPT, could be useful before elective cardiac surgery while LPostC may be considered after acute coronary artery events.