Doctoral Degrees (Medical Physiology)

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Browsing Doctoral Degrees (Medical Physiology) by browse.metadata.advisor "Engelbrecht, Anna-Mart"

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    An investigation into the importance of the ATM protein in the myocardial pathology associated with insulin resistance and type 2 diabetes
    (Stellenbosch : Stellenbosch University, 2017-03) Espach, Yolandi; Huisamen, Barbara; Strijdom, Hans; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology
    ENGLISH ABSTRACT : Introduction: Ataxia-telangiectasia (A-T) is an autosomal, recessive disorder that is caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. The gene product, ATM, is a 350 kDa serine/threonine protein kinase with a large number of substrates in various pathways. Patients suffering from A-T display a high incidence of insulin resistance or type 2 diabetes mellitus (T2DM) and are more susceptible to ischaemic heart disease. Although it is known that the ATM protein is expressed in the heart and that structural and functional changes are observed in the hearts of ATM knock-out mice, very little research has been done on ATM and its role in insulin signalling in the cardiovascular context. This project aimed to identify and characterise an obese, insulin resistant animal model in which myocardial ATM expression is altered; to use this model to determine the effect of ATM manipulation on myocardial function and response to ischaemia/reperfusion injury (IRI); and to determine the effect of ATM manipulation on insulin signalling networks. Methodology: Male Wistar rats received a high caloric diet (HCD) or standard rat chow (control) for 16 weeks after which biometric data was collected. ATM was manipulated in perfusion studies prior to ischaemia using insulin (activator) and KU-60019 (inhibitor). Hearts perfused using the Langendorff balloon model were subjected to global ischaemia followed by reperfusion and Western blots were performed to evaluate insulin signalling intermediates. Hearts were also perfused using the working mode and subjected to regional ischaemia. Following reperfusion, infarct size (IFS) was determined. Aortic ring isometric tension studies were performed to determine the effect of KU-60019 on vasodilation. Results: The HCD resulted in significantly increased body mass, visceral fat mass, glucose levels, insulin levels and HOMA-IR index compared to the control diet and ATM expression was reduced in the HCD hearts. Cardiac function and IFS were comparable in the control and HCD hearts. In control hearts, insulin administration activated the insulin signalling network prior to ischaemia and cardiac function was improved during reperfusion. Insulin had no effect on the insulin signalling network or cardiac function in the insulin resistant HCD hearts. High concentrations of insulin increased IFS in both the control and HCD hearts. ATM inhibition improved cardiac function in control and HCD hearts during early reperfusion but had no effect on cardiac function during later reperfusion. ATM phosphorylation was increased by insulin and decreased by KU-60019 in control hearts, but could not be manipulated in HCD hearts. Insulin-stimulated PKB/Akt activation is not ATM-dependent in the heart. However, ATM inhibition appears to down-regulate insulin signalling via PI3K, PTEN and GSK-3β. ATM inhibition caused NO-dependent vasodilation in control hearts, suggesting a role for ATM in vasoconstriction. Conclusion: ATM is a complex signalling regulator with numerous substrates. In our study, we found that acute cardiac ATM inhibition did not result in significant cardiac dysfunction or complete abrogation of insulin signalling. However, we found that ATM possibly plays a significant role in vasoconstriction. More research needs to be done to fully understand the cardiac role of ATM in insulin signalling.
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    An investigation into the role of ATM protein in mitochondrial defects associated with cardiovascular pathology resulting from insulin resistance
    (Stellenbosch : Stellenbosch University, 2019-04) Blignaut, Marguerite; Huisamen, Barbara; Lochner, Amanda; Engelbrecht, Anna-Mart; Van Vuuren, Derick; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background: Ataxia-telangiectasia (A-T) is a rare, recessive disorder that develops in the absence of Ataxia-Telangiectasia Mutated protein kinase (ATM). This complex disease is characterised by neurodegeneration, increased risk of cancer, a high incidence of insulin resistance and Type 2 diabetes (T2D) as well as cardiovascular disease. Mitochondrial dysfunction is associated with the development of cardiomyopathy and T2D. Obesity and insulin resistance contribute towards the development of cardiac dysfunction, and have been linked with mitochondrial disturbances. ATM has previously been associated with mitochondria, and the absence thereof results in decreased mitochondrial respiration and ATP synthesis as well as structural abnormalities. This study aimed to investigate the role of ATM in mitochondrial oxidative phosphorylation and mitophagy as possible contributors towards cardiovascular dysfunction in obesity. Methodology: The effects of high palmitic and oleic acid, either alone or in combination with insulin or high glucose on ATM expression levels were investigated in an H9c2 cardiomyoblast model. Insulin signal transduction intermediates were determined by western blotting and glucose uptake by [3H]2-deoxyglucose accumulation. Mitoplasts were prepared from cardiac mitochondria of male Wistar rats with digitonin and the quality thereof confirmed with transmission electron microscopy (TEM). The location of ATM was determined with Super-resolution structured illumination microscopy (SR-SIM) and western blotting. Oxidative phosphorylation (oxphos) analysis was performed polarographically (Clark-type electrode) on mitochondria obtained from 1) young male Wistar rat hearts perfused with the ATM specific inhibitor, KU60019 or vehicle (DMSO), and 2) chow fed age-matched controls and diet induced obese (DIO) rat hearts perfused ex vivo with either DMSO or ATM-specific activators and its inhibitor (KU60019 ± insulin or KU60019 ± chloroquine). Oxphos was determined in carbohydrate (glutamate+malate) or fatty acid (palmitoyl-L-carnitine+malate) substrates while protein expression levels of ATM as well as markers of mitophagy and mitochondrial fission were measured (western blotting). The redox status of NAD(P)H was determined with 2-photon fluorescence lifetime imaging microscopy in H9c2 cells ± KU60019. Results: This study showed that: (i) high levels of fatty acids and insulin affects the expression levels of ATM; (ii) ATM is located on the inner mitochondrial membrane of cardiac mitochondria. iii) Inhibition of ATM decreased carbohydrate-stimulated oxidative phosphorylation in cardiac mitochondria (p=0.0024), potentially through Complex I. This was supported by the observation that ATM inhibition decreased NADPH (p=0.02) and increased NADH accumulation in H9c2 cells. iv) The effect of ATM inhibition on oxidative phosphorylation was not influenced by diet; v) ATP synthesis as well as respiratory control index improved with the addition of insulin (p<0.005). vi) The inhibition of ATM was associated with decreased fission (p=0.0038). vii) The decrease in the autophagosomal membrane marker, LC3-II (p<0.0001) seems to be associated with the cytosolic role of ATM. Conclusion: ATM is located on the inner mitochondrial membrane and inhibition thereof influences mitochondrial ATP synthesis, potentially through Complex I substrate oxidation. Inhibition of ATM did not affect oxidative phosphorylation in obesity, but resulted in mitochondrial autophagy disruption as well as decreased fission.
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    Ischaemic preconditioning : an investigation of the patterns of kinase activation and protein expression profiles during reperfusion in the rat heart
    (Stellenbosch : Stellenbosch University, 2013-12) Hattingh, Susanna Maria (Suzel); Du Plessis, Stefan; Engelbrecht, Anna-Mart; Salie, Ruduwaan; Stellenbosch University. Faculty of Medical Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    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.
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    The responses of endothelium to insult : does endothelial heterogeneity play a role in in vitro cell models
    (Stellenbosch : Stellenbosch University, 2015-12) Mthethwa, Mashudu; Strijdom, Hans; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: Endothelial injury and dysfunction precede the development of cardiovascular diseases. The endothelium may be regarded as the first line of defence against inflammation / obesity-induced vascular injury, therefore gaining more information on the mechanisms of injury and response to injury, as well as modulating endothelial function may be key in the prevention of cardiovascular diseases. Endothelial cells differ in structure and function, therefore endothelial heterogeneity may be relevant when investigating endothelial function and dysfunction. Understanding endothelial heterogeneity in response to pathophysiological stimuli may be of significance in the prevention of cardiovascular diseases. Oleanolic acid (OA), a plant-derived triterpenoid, has been shown to possess endothelio-protective properties; however, its role in reversing endothelial injury is poorly understood. This study investigated endothelial heterogeneity between aortic endothelial cells (AECs) and cardiac microvascular endothelial cells (CMECs) at baseline and in response to an inflammatory insult via the cytokine, tumour necrosis factor-alpha (TNF-α). An in vitro model of endothelial injury was developed by treating AECs and CMECs with 20 ng/ml TNF-α for 24 hours. Endothelial heterogeneity was investigated by comparing intracellular nitric oxide (NO) and reactive oxygen species (ROS) production, protein expression and phosphorylation, and large-scale protein expression and regulation in AECs and CMECs. The experimental techniques included flow cytometry, western blots and proteomic analyses. An ex vivo model of endothelial injury was included to investigate vascular function in aortic rings from lean and high fat diet (HFD) rats. The role of OA in reversing TNF-α-induced injury and modulating vascular function in the ex vivo model was investigated. Although baseline NO-levels were similar between AECs and CMECs, heterogeneity was observed with regards to the NO biosynthetic pathway in terms of increased eNOS expression in CMECs. Baseline ROS levels were heterogeneous between AECs and CMECs, interestingly CMECs possessed higher anti-oxidant capacity. An in vitro model of TNF-α-induced injury was confirmed by decreased NO-levels, increased ROS-levels and necrosis, up-regulation of apoptotic proteins and activation of inflammatory pathways in AECs and CMECs. Here, heterogeneity between AECs and CMECs was also observed: endothelial activation was mediated through different proteins in AECs (CD9 molecule, galectin) and CMECs (ICAM-1 and IL-36α). Apoptosis was mediated by caspase 3 in AECs and Bid in CMECs. AECs appeared to advance to a dysfunctional state shown by up-regulation of endothelin-converting enzyme and angiotensin II-converting enzyme, while CMECs maintained an activated state. OA reversed TNF-α-induced injury through restoring NO-production, decreasing ROS-production in both AECs and CMECs, and inhibiting necrosis in AECs. In the ex vivo model of injury, aortic rings from 16-week HFD rats showed a pro-contractile response to phenylephrine-induced contraction, a response that was reversed by OA. In conclusion, we demonstrated novel findings with regards to endothelial heterogeneity between AECs and CMECs under baseline and TNF-α-treated conditions. Although reduced NO-bioavailability may be the hallmark of endothelial dysfunction, signalling pathways mediating endothelial injury may differ between cell types as was shown in this study. We demonstrated that OA possess protective properties in AECs and CMECS, an observation which was translated to the ex vivo model.
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    The role of Protein Phosphatase 2A (PP2A) in myocardial ischaemia/reperfusion injury
    (Stellenbosch : Stellenbosch University, 2014-04) Van Vuuren, Derick; Lochner, Amanda; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Division of Medical Physiology.
    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.