Thesis (MScMedSc (Dept. of Biomedical Sciences. Medical Physiology))University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Background: Obesity is associated with dyslipidemia, insulin resistance and glucose intolerance and together these components characterise the metabolic syndrome (Dandona et al. 2005). In the state of obesity, there are high levels of circulating free fatty acids and increased rates of fatty oxidation which inhibit glucose oxidation. This: (i) reduce the heart‘s contractile ability, (ii) exacerbates ischemic/reperfusion injury and (iii) decreases cardiac mechanical function during reperfusion (Kantor et al. 2000; Liu et al. 2002; Taegtmeyer, 2000). Aim: The aim of our study was to investigate the effect of inhibiting fatty acid oxidation, with oxfenicine (4-Hydroxy-L-phenylglycine), on (i) cardiac mechanical function, (ii) mitochondrial respiration, (iii) myocardial tolerance to ischemia/reperfusion injury, (iv) CPT-I expression, MCAD expression, IRS-1 activation, total GLUT- 4 expression and (v) the RISK pathway (ERK42/44 and PKB/Akt). Methods: Male Wistar rats were fed a control rat chow diet or a high calorie diet (HCD) for 16 weeks. The HCD caused diet induced obesity (DIO). The animals were randomly divided into 4 groups [Control, DIO, Control + oxfen and DIO + oxfen]. The drug was administered for the last 8 weeks of feeding (200mg/kg/day). Animals were sacrificed and the hearts were perfused on the Langendorff perfusion system. After being subjected to regional ischemia and two hours of reperfusion, infarct size was determined. A separate series of animals were fed and/or treated and hearts were collected after 25 minutes global ischemia followed by 30 min reperfusion for determination of GLUT- 4, CPT-1, IRS -1, MCAD, ERK (42/44) and PKB/Akt expression/phosphorylation using Western blot analysis. A third series of hearts were excised and used for the isolation of mitochondria. Results: In the DIO rats, chronic oxfenicine treatment improved cardiac mechanical function by improving mitochondrial respiration. Oxfenicine inhibited CPT-1 expression but had no effect on MCAD or GLUT- 4 expression. Oxfenicine decreased IRS-1 iv expression, but not IRS-1 activation. Oxfenicine also improved myocardial tolerance to ischemia/reperfusion without activation of the RISK pathway (ERK & PKB). In the control rats, chronic oxfenicine treatment worsened cardiac mechanical function by adversely affecting mitochondrial respiration. Oxfenicine also worsened myocardial tolerance to ischemia/reperfusion in the control rats without changes in the RISK pathway (ERK & PKB). Oxfenicine had no effect on CPT-1, MCAD or GLUT- 4 expression. Oxfenicine increased IRS-1 expression, but not IRS-1 activity. Conclusion: Chronic oxfenicine treatment improved cardiac mechanical function and myocardial resistance to ischemia/reperfusion injury in obese animals, but worsened it in control animals. The improved cardiac mechanical function and tolerance to ischemia/reperfusion injury may be due to improvement in mitochondrial respiration. / AFRIKAANSE OPSOMMING: Agtergrond: Vetsug word geassosieer met dislipidemie, insulien weerstandigheid en glukose intoleransie, wat saam die metaboliese sindroom karakteriseer (Dandona et al. 2005). Met vetsug is daar ‗n hoë sirkulasie van vetsure, sowel as verhoogde vertsuur oksidasie wat gevolglik glukose oksidasie onderdruk. Dit: (i) verlaag die hart se vermoë om saam te trek, (ii) vererger isgemiese/herperfusie skade en (iv) verlaag kardiale effektiwiteit gedurende herperfusie (Kantor et al. 2000; Liu et al. 2002; Taegtmeyer, 2000). Doel: Die doel van die studie was om die effekte van vetsuur onderdrukking m.b.v. oksfenisien (4-Hidroksie-L-fenielglisien) op (i) meganiese hart funksie, (ii) mitokondriale respirasie, (iii) miokardiale toleransie teen isgemiese/herperfusie skade, (iv) CPT-I uitdrukking, MCAD uitdrukking, IRS-1 aktiwiteit, totale GLUT-4 uitdrukking en (v) die RISK pad (ERK42/44 en PKB/Akt) te ondersoek. Metodes: Manlike Wistar rotte was gevoer met ‗n kontrole rot dieet of ‗n hoë kalorie dieet (HKD) vir 16 weke. Die HKD lei tot dieet-geïnduseerde vetsug (DGV). Die diere was lukraak verdeel in 4 groepe [kontrole, DGV, kontrole + oksfen en DGV + oksfen]. Die behandeling met die middel was toegedien vir die laaste 8 weke van die voeding protokol (200mg/kg/dag). Die diere was geslag en die harte was geperfuseer op die Langendorff perfusie sisteem. Na blootstelling aan streeks- of globale isgemie en 2 ure herperfusie was infark groottes bepaal. ‗n Aparte reeks diere was gevoer en/of behandel en die harte was versamel na 25 minute globale isgemie gevolg deur 30 minute herperfusie vir die bepaling van GLUT-4, CPT 1, IRS -1, MCAD, ERK (42/44) en PKB/Akt uitdrukking/aktivering d.m.v. Western blot analise. ‗n Derde reeks diere was gebruik vir die isolasie van mitokondria. Resultate: In die DGV diere, het kroniese oksfenisien behandeling meganiese hart funksie verbeter d.m.v. die verbetering van mitokondriale respirasie. Oksfenisien het CPT-1 uitdrukking verlaag terwyl GLUT- 4 en MCAD uitdrukking nie geaffekteer was vi nie. Oksfenisien het IRS-1 uitdrukking verlaag, maar nie IRS-1 aktiwiteit nie. Oksfenisien het ook miokardiale weerstand teen isgemiese/herperfusie verbeter met sonder aktivering van die RISK pad (ERK & PKB). In die kontrole diere, het kroniese oksfenisien behandeling die meganiese hart funksie versleg d.m.v. negatiewe effekte op mitokondriale respirasie. Oksfenisien het die miokardiale weerstand teen isgemiese/herperfusie van die kontrole rotte versleg sonder veranderinge in die RISK pad (ERK & PKB). Oksfenisien het geen effek gehad op CPT-1, MCAD en GLUT-4 uitdrukking nie. Oksfenisien het IRS-1 uitdrukking verhoog, maar nie IRS-1 aktiwiteit nie. Samevatting: Kroniese oksfenisien behandeling het die meganiese hart funksie en miokardiale weerstand teen isgemiese/herperfusie skade in die vet diere verbeter, maar versleg in die kontrole diere. Hierdie verbetering van meganiese hart funksie en weerstand teen isgemiese/herperfusie skade kon dalk wees a.g.v. ‗n verbetering in mitokondriale respirasie.

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.