Renal ischaemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) which is in turn the leading cause of morbidity and mortality in hospitalized patients. The principle aim of this thesis was to evaluate potential new therapies that might afford protection against IRI in both in vitro and in vivo settings. Recent evidence suggests that nitrite (NO2-) may play an important role in protecting the myocardium from IRI. Our initial work into the role of NO2- in an in vitro model of renal IRI in proximal tubular epithelial cells provided evidence that NO2- can prevent apoptosis and preserve cell viability. This lead to an in vivo study where high NO2- concentrations (50 mg/L) were given orally to rats for 7 days prior to inducing renal IRI but no beneficial effects of this treatment were observed. Another potential treatment identified was thiamine (vitamin B1) and this, like NO2-was investigated to see if it had the potential to protect rats from AKI injury. It has been previously recognized that in renal IRI the high energy phosphate ATP is found to be severely depleted whilst is is known that thiamine can play a pivotal role in generating ATP. Furthermore, thiamine has previously been demonstrated to protect against myocardial ischaemic injury and has the ability to reduce myocardial infarct size. In vitro, thiamine was found to reduce the degree of apoptosis in cultured HK-2 cells caused by ischaemia whilst in vivo it afforded protection against AKI caused by renal IRI by anti-apoptotic, anti-inflammatory and anti-oxidant mechanisms. Finally, a study into the possible therapeutic role of gene therapy with bone morphogenic protein 7 (BMP-7) in renal IRI was undertaken. Previous work has established that i.v. BMP-7 is able to protect against renal IRI but it has also been associated with ectopic bone formation at the site of injection. Therefore another method to increase circulating BMP-7 was sought. We initially found that BMP-7 gene therapy could attenuate apoptosis and preserves cell viability in an in vitro model of renal IRI. However, whilst in vivo gene therapy with electroporation of BMP-7 plasmid DNA increased BMP-7 expression in mice serum 2 days post electroporation, it was unable to protect the animals against IRI induced AKI. In rats the direct injection of naked DNA BMP-7 plasmid systematic 2 days prior to renal IRI was able to upregulate BMP-7 expression 4 days later in kidney tissue. Despite this it was unable to afford protection against renal IRI. Apoptosis and necrosis play a crucial role in the pathogenesis of renal IRI induced AKI. In this thesis we investigated the role of three putative therapeutic agents and their role in apoptosis and necrosis in vitro in PTECs and in vivo against renal IRI induced AKI. All three therapeutic drugs were able to attenuate apoptosis in PTECs but were unable to protect against necrosis, whilst against renal IRI induced AKI only thiamine was found to be protective. Thiamine appears to hold the most promise and more work needs to be undertaken so that its potential benefit in AKI can be realised.

Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation and increases the risks of primary non-function, delayed graft function and rejection. Oxidative damage to mitochondria is a key early event in IR injury. The aim of this project was to examine the safety and efficacy of the mitochondria-targeted antioxidant MitoQ in reducing pig and human kidney IR injury using an ex vivo normothermic perfusion (EVNP) system. Over a range of 500 nM to 250 µM using a 150 pig kidneys and 80 declined deceased human kidneys, MitoQ was successfully taken up by pig and human kidneys in a concentration-dependent manner, resulting in stable tissue concentrations over 24 hours of cold storage followed by 6 hours of EVNP. The uptake of MitoQ was increased approximately 2-fold when MitoQ was administered to warm (rather than cold) kidneys and when kidneys were preserved using hypothermic machine perfusion (rather than cold static storage). 50 µM MitoQ, administered to pig kidneys at the end of warm ischaemia, significantly increased renal blood and urine output flow at the end of 6 h EVNP compared to the control group. Creatinine clearance was numerically higher in the 50 µM MitoQ group compared to the control group but the difference did not reach statistical significance. To test the safety and efficacy of MitoQ in human kidney IRI, pairs of declined deceased human kidneys were used, with one kidney in each pair used as control. The total urine output, creatinine clearance and percentage fall of serum creatinine were numerically higher in the 50 µM MitoQ group compared to the control group, although the differences did not reach statistical significance during 3 h of EVNP. There was a significant difference in the renal blood flow between the 50 µM MitoQ group and the control group at the end of the first hour of EVNP. The renal blood flow remained relatively stable during the first hour of EVNP in the 50 µM MitoQ group compared to a significant decrease in renal blood flow in the control group. There was no effect on fractional excretion of sodium or oxidative injury markers (protein carbonyl formation, lipid peroxidation) in pig or human kidneys, which is consistent with previous studies that demonstrated the requirement of >24 hour after reperfusion for manifestation of changes in these parameters. In this thesis, I was able to successfully demonstrate the safety and potential efficacy of MitoQ in ameliorating renal IRI using pig kidneys. While more declined deceased human kidneys need to be analysed to fully explore the potential efficacy of MitoQ in ameliorating renal IRI, this study provides important data that will help inform future studies and ultimately a clinical trial for assessing the efficacy of the mitochondria-targeted antioxidant MitoQ in human kidney transplantation. My findings suggest that MitoQ has the potential to increase the use of marginal kidneys and to improve graft and patient outcomes.

Aims There are few proven therapies that can protect against the inevitable ischaemia reperfusion injury (IRI) that occurs during renal transplantation. IRI increases the likelihood of delayed graft function (DGF), which negatively impacts on the long-term survival of a transplanted kidney. One enzyme of interest, heme oxygenase-1 (HO-1), degrades heme and protects against the oxidative stress that occurs secondary to IRI. Clinical renal recipients with higher HO-1 levels have improved graft function post transplant. Heme arginate (HA), a form of hemin, which has been used to treat porphyria for over 30 years, has repeatedly been shown to induce HO-1 in in vivo and in vitro macrophages. It is one of the few HO-1 inducers approved for clinical use and healthy volunteer studies confirmed that HA could also safely induce HO-1 in humans. Prior to the formal start of the MD, the University of Edinburgh successfully applied to NHS Blood and Transplant for funding to investigate whether giving HA to recipients of deceased donor renal grafts prior to transplant could upregulate HO-1 and whether this had any effect on the function and health of the grafts. This MD aims to explain the background behind the proposed study, the process of study approval, planning and trial logistics and protocol. This thesis then describes the methods of sample analysis, the results and future directions for the HOT (Heme Oxygenase-1 in renal Transplantation) study. Methods The HOT study planning and approval process took eight months and the first participant was randomised in January 2012. The study was sponsored by ACCORD, a joint company from University of Edinburgh and NHS Lothian, and recruited patients from the Edinburgh Royal Infirmary Transplant Unit. The protocol was followed to ensure that 40 recipients were randomised blind to either active (two doses 3mg kg-1 HA: pre-operatively, day 2) or placebo (NaCl: same schedule). To ensure that the primary outcome was fulfilled, recipient blood was taken daily for peripheral blood mononuclear cells (PBMC) extraction. After further blinding steps, the PBMCs were analysed for HO-1 protein and mRNA. The secondary outcome measures involved collecting urine for analysis of urinary biomarkers (KIM-1 and NGAL), taking renal graft biopsies pre-op and day 5 for renal HO-1 analysis and collecting renal function data. DGF was calculated daily. To ensure that all adverse event data was captured, the recipients were closely reviewed for 7 days and their renal function was monitored for 90 days. Results The final participant was recruited in May 2013 within the predicted timescale and to budget. This participant completed follow-up in August 2013. Of the 40 participants, three received the infusion but did not receive a transplant and therefore could not give primary outcome data. The remaining 37 did and this was analysed. Adverse events were equivalent between groups and there were no adverse reactions to HA. HA upregulated PBMC HO-1 protein at 24 hours compared to placebo: HA 11.1ng/ml (1.0- 37.0) vs. placebo 0.14ng/ml (-0.7- 0.3)(p= < 0.0001). PBMC HO-1 mRNA was also increased: HA 2.73 fold (1.8- 3.2) vs. placebo 1.41 fold (1.2- 2.2) (p=0.02). HA increased HO-1 protein immunopositivity in day 5 renal tissue compared with placebo: HA 0.21 (-24- 0.7) vs. placebo -0.03 (-76- 0.15) (p=0.02) and the percentage of HO-1 positive renal macrophages also increased: HA 50.8 cells per HPF (40.0- 59.8) vs. placebo 22.3 (0- 34.8) (p=0.012). Renal HO-1 mRNA was also increased in HA group: 2.02 (0.20- 4.03) fold increase compared to 1.68 (0.75- 10.39) fold in the placebo group but it was not significant (p= 0.451). Urinary biomarkers were reduced after HA but not significantly so. Histological injury and DGF rates were similar between the groups. Conclusion HA is safe and effective in renal transplant recipients as reported in this phase II, randomised, placebo controlled, blinded, single-centre study. The primary outcome was achieved and demonstrated for the first time that HA induces HO-1 in peripheral and renal macrophages in kidney transplant recipients. There was also evidence that HA increased HO-1 expression in renal tissue. There was no evidence that HA improved renal function or reduced injury as seen in animal models but it is recognised that the sample size was small and the study was not powered to these endpoints. Larger studies are planned to determine the impact of HO-1 upregulation on clinical outcomes and evaluate the benefit to patients at risk of IRI. The plans for HOT2 are expanded in this thesis.

Brain death has adverse effects on the organ donor, increasing organ dysfunction and affecting transplantation outcomes. It can also render organs unsuitable for transplantation. Another determinant of organ quality is ischaemia-reperfusion injury, which limits ischaemic storage time for hearts to six hours. The aim of this thesis was to investigate the effectiveness of hormone resuscitation (HR) of the donor to ameliorate the effects of brain death. Another aim was to develop a donor management and organ preservation strategy to ameliorate the effects of ischaemia-reperfusion injury on the heart, thereby extending ischaemic preservation times. A porcine model of the brain-dead multi-organ donor with orthotopic cardiac transplantation was utilised. Donor HR was shown to improve cardiac contractility and haemodynamics, thereby reducing inotrope requirements. A follow-up study investigating the effects of three different donor management protocols demonstrated that donor haemodynamics, renal arterial flow and creatinine clearance were superior in HR animals compared with animals treated with noradrenaline or intravenous fluid alone. Noradrenaline was associated with a significant deterioration in pulmonary function (PaO2 and alveolar-arterial oxygen gradient) and a decline in donor pH. HR was not associated with any detrimental effects on the lungs, liver or pancreas compared with the other two groups. Preservation strategies incorporating glyceryl trinitrate (GTN) and cariporide, a Na+-H+ exchange inhibitor, were investigated to safely extend cardiac ischaemic preservation times. Pre-treatment with intravenous cariporide prior to heart explantation (donor) and reperfusion of the transplanted heart (recipient) was shown to effectively extend ischaemic time to 14 hours, evidenced by weaning off cardiopulmonary bypass. GTN and cariporide-supplemented Celsior, used as a cardioplegic/storage solution, was also effective in extending preservation time to 14 hours, with superior cardiac contractility compared with cariporide pre-treated hearts. Both treatments also ameliorated reperfusion injury, stabilising haemodynamics for up to three hours post-bypass. This thesis has demonstrated the effectiveness of HR to ameliorate the negative effects of donor brain death. It also provides evidence that combined GTN and cariporide-supplemented Celsior improves long-term preservation of the donor heart. These strategies offer the potential to increase the proportion of transplantable organs, to improve donor organ quality, and thereby improve transplantation outcomes.

Background: Glucagon-like Peptide 1 (GLP-1) is a human incretin hormone that has been demonstrated to protect against non-lethal ischaemia reperfusion injury in the left ventricle in humans. It has been suggested from some animal research that this protection may be mediated through the pathway of ischaemic conditioning, of which the opening of the mKATP channel is a key step. Furthermore, it is uncertain whether the protection applies to the right ventricle. Finally, there is limited human evidence of a protective effect against lethal ischaemia reperfusion injury. Methods: Two studies use non-lethal ischaemia to test whether GLP-1 protection is maintained despite blockade of the mKATP channel with the sulfonylurea, glibenclamide. A demand ischaemia study uses dobutamine stress echo to compare LV function. The other uses transient coronary balloon occlusion to generate supply ischaemia during GLP-1 infusion, assessed by conductance catheter. A further transient balloon occlusion is also used to assess the effect of supply ischaemia on RV function. Finally, the GOLD PCI study assesses whether GLP-1 protects against periprocedural myocardial infarction when administered during elective PCI in a randomised, placebo controlled double blind trial. Results: Glibenclamide did not affect GLP-1 cardioprotection in either supply of demand ischaemia suggesting that GLP-1 protection is not mediated through the mKATP channel. The RV experienced stunning with RCA balloon occlusion but there was little evidence of cumulative ischaemic dysfunction with further occlusions. GOLD PCI is continuing to recruit patients. The nature of the study means results cannot be assessed until recruitment is complete. Conclusions: GLP-1 is an agent with potential for clinical use as a cardioprotective therapy. It’s mechanism of action in the heart remains uncertain.

Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors that mediate various actions of Acetylcholine (ACh) in the central nervous system and peripheral nervous system. In mammals, five distinct mAChR subtypes (M1-M5) have been recognised with the M2 subtype being predominantly present in the heart. The mAChR antagonists are routinely used for the treatment of various pathophysiological conditions including respiratory conditions. However, it has been postulated that mAChR antagonists may increase morbidity and mortality in chronic obstructive pulmonary disorder (COPD) and asthma patients with underlying cardiovascular disease, raising concerns regarding the cardiovascular safety of these agents. The current study was therefore undertaken to investigate the effects of individual mAChR antagonists in the setting of myocardial ischaemia reperfusion injury and oxidative stress models. We also investigated whether the inhibition of the mitochondrial permeability transition pore (MPTP) with cyclosporine-A (CsA) in the presence and absence of individual mAChR antagonists provided protection against ischaemia reperfusion injury. Furthermore, we also aimed to investigate the intracellular signalling pathway associated with mAChRs antagonists mediated myocardial injury under the stress conditions. Langendorff results showed that the non-selective M1-M3 mAChR antagonist, ipratropium bromide, the M2 mAChR antagonist, AF-DX 116 and the M3 mAChR antagonist, DAU 5884 significantly increased the infarct size to risk ratio of the heart in conditions of ischaemia and reperfusion. Detrimental effects of AF-DX 116 and DAU 5884 were abrogated by co-treatment of these drugs with mAChR agonist, acetylcholine (ACh) and/or CsA. Cell viability data of isolated cardiac myocytes revealed that AF-DX 116 and DAU 5884 caused a concentration dependent decrease in the viability of cardiac myocytes as well as causing a reduction in the time taken to depolarisation and hypercontracture under oxidative stress. AF-DX 116 and DAU 5884 significantly increased the levels of p-SAPK/JNK and decreased the levels of p-Akt and p-ERK. In addition, ACh and CsA showed to activate p-Akt and p-ERK. To conclude, the data suggest that AF-DX 116 and DAU 5884 caused cardiotoxicity at cellular, tissue and protein level in conditions of ischaemia reperfusion injury and oxidative stress. Furthermore, inhibition of the mitochondrial transition pore with CsA protected against the AF-DX 116 and DAU 5884 induced injury via activation of the pro-survival proteins, p-Akt and p-ERK.

Aims: Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BARΔRING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity. Methods and results: We generated a line of transgenic mice that carried a human BARΔRING transgene under the control of the mouse α-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BARΔRING transcripts and 42 KDa BARΔRING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 ± 1.7% in the non-transgenic hearts compared with 51.5 ± 4.3% in the transgenic hearts (P < 0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 ± 4 vs. 55 ± 4%, P < 0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 ± 0.4 vs. 13.4 ± 0.5%, P < 0.05). Over-expression of BARΔRING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis. Conclusion: Our results demonstrate that over-expression of BARΔRING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Obesity has reached epidemic proportions worldwide and is currently a serious health problem. It is associated with metabolic abnormalities, oxidative stress, hypertension, insulin resistance and an increased disposition for the development of cardiovascular disease. Elucidation of the pathophysiological mechanisms underlying obesity and its relationship with metabolic and cardiovascular diseases is essential for prevention and management of these disorders. Melatonin, the pineal gland hormone, is a powerful antioxidant and has been shown to protect the myocardium against ischaemia/reperfusion (I/R) injury. Long- as well as shortterm melatonin treatment also reversed several of the harmful effects of obesity in an animal model of hyperphagia-induced obesity (DIO). However, its effects on myocardial I/R injury and intracellular signalling in obesity induced by a high fat diet (HFD) are still unknown. Aims of study: (i) To evaluate the ability of a high fat diet (HFD) to induce obesity in rats. Apart from evaluating its effects on the biometric parameters and resistance to ischaemia/reperfusion injury (as indicated by infarct size in regional ischaemia and functional recovery after global ischaemia), special attention will be given on the interplay between adiponectin, AMPK, leptin, and FFA in this model. (ii) To evaluate the effect of daily oral administration of melatonin to rats on the HFD as well as their littermate controls, on the parameters listed above as well as on the development of obesity. In this study melatonin will be administered from the onset of the feeding of the high fat diet. Methods: Male Wistar rats were divided into 4 groups: (i) control rats (receiving normal rat chow) (C); (ii) control rats receiving melatonin (CM); (iii) obese rats (receiving HFD) (HFD); (iv) obese rats receiving melatonin (HM). Animals were kept on the diet for 16 weeks and melatonin treatment (10mg/kg/day, added to the drinking water) started at the onset of the feeding. Following feeding and treatment, the animals were grouped into fasted/ non-fasted of which biometric parameters were recorded and blood collected at the time of sacrifice for metabolic and biochemical assays. Hearts were perfused in the working mode for evaluation of myocardial function and infarct size determination after exposure to 35min regional ischaemia/60min reperfusion. For study of intracellular signaling, hearts were perfused in the working mode, subjected to 20min global ischaemia/10min reperfusion and freeze-clamped for Western blotting. Plasma leptin, adiponectin, free fatty acid, triglycerides, total cholesterol, phospholipids, conjugated dienes and thiobarbituric reactive substances (TBARS) levels were determined. Several kinases were investigated including, the RISK (reperfusion injury salvage kinase) (PKB/Akt and ERK p44/42) and SAFE (survivor activating factor enhancement) (STAT-3) pathways, AMPK and JNK under baseline conditions or following 10 min reperfusion. In addition, expression of UCP-3 and PGC1-α was determined. Results: Significant increases in body weight, visceral fat, blood glucose, insulin, HOMA index and leptin and a reduction in adiponectin levels were observed in the fasted high fat diet (HFD) group when compared with controls (C). Significant increases in free fatty acid and triglyceride levels were also noted the HFD group while other serum lipid parameters, including TBARS, remained unchanged. No differences in functional recovery during reperfusion or infarct size after exposure to 35 min regional ischaemia, as well as functional recovery during reperfusion after 20 min global ischaemia were observed between the control and HFD groups. Baseline and 10 min reperfusion data were similar for the RISK and SAFE pathway kinases for the control vs HFD groups. The HFD also had no effect on the expression and phosphorylation of myocardial AMPK and JNK, as well as on the expression of UCP-3 and PGC1-α, when compared to the controls. Treatment with melatonin significantly reduced body weight, visceral fat, blood glucose, HOMA index and serum leptin levels in HFD treated groups, while having no effect on the lipid profile. Although melatonin significantly reduced infarct size in both control [% of area at risk: 20.59 ± 2.29 (CM) vs 38.08 ± 2.77 (C)] and high-fat diet groups [% of area at risk: 11.43 ± 2.94 (HM) vs 38.06 ± 3.59 (H)], it was without effect on myocardial functional recovery during reperfusion. Melatonin had no effect on the intracellular signaling pathways studied. Conclusions: The HFD proved to be a useful model of diet-induced obesity with a more pronounced impact on biometric and metabolic changes compared to the DIO model. Long-term melatonin treatment successfully prevented the development of metabolic abnormalities associated with the high fat diet and obesity as well as significantly reduced myocardial infarct size. The mechanisms involved in melatonin-induced cardioprotection in obesity have not been fully elucidated in this study and require further investigation. However, the anti-obesogenic and cardioprotective properties of melatonin were very significant indeed and support the suggestion of this hormone as a potential tool in the treatment of obesity and associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding: Vetsug (obesiteit) het wêreldwyd epidemiese afmetings aangeneem en word tans as ‘n ‘n ernstige gesondheidsprobleem beskou. Vetsug word geassosieer met metaboliese afwykings, oksidatiewe stres, hipertensie, insulienweerstandigheid en is‘n belangrike risikofaktor vir die ontwikkeling van kardiovaskulêre siekte. Ten spyte hiervan, het onlangse studies ‘n gunstige effek van vetsug op die uitkomste van miokardiale infarksie in pasiënte gerapporteer, die sg obesiteitsparadoks. Kennis van die patofisiologiese meganismes onderliggend aan vetsug en die ontstaan van metaboliese afwykinge en hartsiekte is noodsaaklik vir die voorkoming en behandeling van hierdie toestande. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige antioksidant en vry radikaal opruimer. Dit is voorheen aangetoon dat dit die hart teen iskemie/herperfusie (I/H) besering kan beskerm en sommige van die skadelike gevolge van vetsug in diermodelle kan omkeer. Die effek van melatonien op miokardiale I/H besering en intrasellulêre seintransduksie prosesse in vetsug geïduseer deur ‘n hoë vet dieet is egter nog onbekend. Doelstellings: (i) Die ontwikkeling en karakterisering van ‘n nuwe model van vetsug en insulienweerstandigheid geïnduseer deur 'n hoë vet dieet (HVD) en die evaluering van die effek daarvan op miokardiale I/H besering en die gepaardgaande intrasellulêre seintransduksieprosesse; (ii) Bepaling van die effek van daaglikse toediening van melatonien aan rotte op die HVD sowel as aan kontroles op ‘n standard dieet, op die ontwikkeling van dieet-geïnduseerde metaboliese veranderinge, miokardiale infarktgrootte en funksionele herstel na koronêre arterie afbinding, sowel as intrasellulêre seintransduksie. Metodiek: Vier groepe van manlike Wistar rotte is bestudeer: (i) kontrole rotte (op‘n standaard dieet) (K); (ii) kontrole rotte op ‘n standard dieet plus melatonien (KM); (iii) dieetrotte (op‘n HVD); (iv) HVD rotte wat melatonien ontvang (HM). Die HVD en melatonien (10mg/kg/dag in die drinkwater) is vir 16 weke toegedien. Na die periode van behandeling, is die diere in vastende en nie-vastende groepe verdeel, die biometriese parameters genoteer en bloedmonsters vir metaboliese en biochemiese bepalings versamel, tydens verwydering van die harte. Harte is geperfuseer volgens die werkhartmodel vir bepaling van miokardiale funksie en infarktgrootte na blootstelling aan 35min streeksiskemie. Vir evaluering van intrasellulêre seintransduksie, is geperfuseerde werkende rotharte blootgestel aan 15min globale iskemie/10 min herperfusie en gevriesklamp vir latere analises volgens die Western kladtegniek.hart. Serum leptien, adiponektien, vryvetsure, trigliseried, totale cholesterol, fosfolipiede, gekonjugeerde diene en tiobarbituursuur reaktiewe stowwe (TBARS) is bepaal. Met gebruik van Western kladtegniek, is die aktivering en/of uitdrukking van die RISK (PKB/ Akt en ERK p44/42) en SAFE (STAT-3) seintransduksiepaaie, AMPK, JNK, UCP-3 en PGC1-α, onder basislyn toestande of na 10 min herperfusie bestudeer. Resultate:‘n Beduidende toename in liggaamsgewig, visserale vet, die HOMA indeks, insulien en leptien vlakke is in die HVD groep waargeneem vergeleke met die kontrole (K) rotte. Adiponektien vlakke was laer in die HVD groep. Die HVD groep is ook gekenmerk deur ‘n beduidende styging in serum vryvetsuur en trigliseried vlakke, terwyl die ander lipied parameters, insluitende die TBARS vlakke, onveranderd was. Infarktgrootte en funksionele herstel tydens herperfusie na blootstelling aan 35 min streeksiskemie, asook funksionele herstel tydens herperfusie na 20 min globale iskemie het nie verskil tussen harte van die kontrole en HVD rotte nie. Aktivering van PKB/Akt, ERK p44/p42, STAT3, AMPK en JNK by basislyn en na 10 min herperfusie was soortgelyk in die kontrole en HFD groepe. Die HVD het ook geen effek op die uitdrukking van UCP-3 en PGC1-α in vergelyking met die kontrole gehad nie. Behandeling met melatonien het die liggaamsgewig, visserale vet, bloedglukose, HOMA indeks en serum leptien vlakke in die HVD groepe statisties beduidend verlaag, terwyl dit geen invloed op die lipiedprofiel gehad het nie. Melatonien behandeling het die miokardiale infarktgrootte beduidend en tot dieselfde mate verminder in beide kontrole [20.59 ± 2.29 (KM) vs 38.08 ± 2.77% (K)] en HVD groepe [11.43 ± 2.94 (HM) vs 38.06 ± 3.59% (HVD)]. Geen verskille is egter tussen die funksionele herstel gedurende herperfusie van die behandelde en onbehandelde kontrole en HVD groepe waargeneem nie. Melatonien het ook geen uitwerking op die intrasellulêre seintransduksiepaaie gehad nie. Gevolgtrekkings: Die resultate het getoon dat die HFD 'n goeie model van dieetgeïnduseerde vetsug en insulien weerstandigheid ontlok, met 'n meer uitgesproke impak op biometriese en metaboliese veranderinge as die voorheen gebruikte hoë-sukrose dieet. Langtermyn melatonien- behandeling het die ontwikkeling van metaboliese abnormaliteite geassosieer met die HVD, voorkom, asook miokardiale infarktgrootte na koronêre afbinding beduidend verminder. Die meganismes betrokke in melatonien-geïnduseerde miokardiale beskerming moet egter in meer detail ondersoek word. Die resultate verkry steun die voorstel dat melatonientoediening voordelig sal wees in die behandeling van vetsug en sy kardiovaskulêre komplikasies.

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.