Investigating aberrant DNA methylation patterns in LOXL2 as a potential predictor of diabetes-induced cardiac dysfunction
| dc.contributor.advisor | Johnson, Rabia | en_ZA |
| dc.contributor.advisor | Samodien, Ebrahim | en_ZA |
| dc.contributor.advisor | Maarman, Gerald | en_ZA |
| dc.contributor.advisor | Pheiffer, Carmen | en_ZA |
| dc.contributor.author | Erasmus, Melisse | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. | en_ZA |
| dc.date.accessioned | 2021-06-06T11:14:30Z | |
| dc.date.accessioned | 2021-12-22T14:11:01Z | |
| dc.date.available | 2021-06-06T11:14:30Z | |
| dc.date.available | 2021-12-22T14:11:01Z | |
| dc.date.issued | 2021-04 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Cardiovascular disease (CVD) remains the leading cause of mortality worldwide, with 17.9 million deaths annually. CVD pathophysiology is often characterized by increased stiffening of the heart muscle due to fibrosis, thus resulting in diminished cardiac function. Fibrosis can be caused by increased oxidative stress and inflammation, which is strongly linked to lifestyle and the environment. Diets rich in fat and sugar have been implicated as one of the prominent risk factors for the occurrence of CVD including associated risk factors such as obesity and insulin resistance. These diets are known to induce an inflammatory response that promotes myocardial stiffening due to increased cardiac fibrosis. Recently, Lysyl oxidase-like 2 (LOXL2), an enzyme required for collagen and elastin cross-linking in the myocardium, has been proposed to have a profound role in cardiac fibrosis and mechanical dysfunction of stressed hearts. Despite acknowledging the crucial role of LOXL2 as an early marker of cardiac fibrosis, there is a paucity of data on LOXL2 regulation and its role in CVD. Additionally, there is a definite need to investigate if LOXL2 regulation may be mediated by epigenetic mechanisms. This would greatly aid in identifying a means to control and manipulate LOXL2, particularly in humans, thereby developing novel therapeutics to combat this worldwide CVD problem. As such, this study aims to gain insight into (1) the mechanisms by which LOXL2 and its downstream effectors are regulated in physiological conditions responsible for CVD development, (2) whether LOXL2 expression in male and female rats is regulated by DNA methylation, and (3) if a pharmaceutical grade of green rooibos (AfriplexGRTTM) can prevent altered gene expression. Furthermore, in search of new therapeutic targets, this study also aimed to investigate the ameliorative effects of AfriplexGRTTM on high glucose and palmitate (HG+PAL)-induced stress in an in vitro H9c2 and in vivo obesogenic diet-induced cardiac fibrosis using a Wistar rat model. Methods: For the in vitro model, H9c2 cells were treated with HG+PAL for 24 hours, followed by further 6-hour treatment with AfriplexGRTTM, or its bioactive compound Aspalathin. The effect of stress induction was investigated by studying ATP production, ROS generation, mitochondrial bioenergetics, mitochondrial membrane potential and apoptosis. Cells were also harvested for mRNA expression analysis. To establish a model of diet-induced fibrosis in vivo, Wistar rats were fed different obesogenic diets, including a high-fat, high-sugar (HFHS) diet, and obesogenic 1 (OB1) diet, or an obesogenic 2 diet (OB2), over a set period with or without supplementation with AfriplexGRTTM. Bodyweight, food intake and fasting blood glucoses were monitored over the study period, after which blood was collected for serum analysis of lipids, triglycerides, insulin levels as well as serum LOXL2 levels. Heart tissue was collected for DNA methylation profile (HFHS diet only), mRNA, and protein expression analysis. Results: Results from the in vitro study showed that HG+PAL treatment induced an inflammatory response, increased oxidative stress and reduced mitochondrial membrane potential, increased apoptosis. It, however, could not induce a significant change in gene expression levels of various fibrotic markers including LOXL2. AfriplexGRTTM and Aspalathin treatments were unable to ameliorate the detrimental effects induced by HG+PAL treatment. Results from the in vivo studies indicated that the obesogenic diets were able to increase animal bodyweights, serum insulin levels, triglyceride levels and serum LOXL2, with no significant differences were observed at a gene expression or DNA methylation level. However, enhanced protein expression levels correlated with increased serum levels of LOXL2 in male Wistar rats. Therefore, it has been postulated that early events of cardiac fibrosis might have been activated, that require further validation in future studies. Conclusion: This study demonstrated that an obesogenic diet can induce alterations in metabolic parameters that are gender-specific. These findings also indicated that LOXL2 regulation may have been driven by a mechanism independent of DNA methylation and that AfriplexGRTTM was unable to mitigate LOXL2-induced fibrosis or cardiac perturbations in these diet-induced obesity Wistar rat models. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Kardiovaskulêre siektes (KVS) bly die grootste oorsaak van dood wêreldwyd, met 17.9 miljoen sterftes jaarliks. KVS patofisiologie word dikwels gekenmerk deur verhoogde verstywing van die hartspier as gevolg van fibrose, wat lei tot verswakte hartfunksie. Fibrose kan deur verhoogde oksidatiewestres en inflammasie veroorsaak word, wat gekoppel is aan leefstyl en omgewingsfaktore. Diëte met hoë vet- en suikerinhoud was voorheen geïdentifiseer as een van die hoof risikofaktore vir die voorkoms van KVS, insluitend verwante siektes soos vetsug en insulienweerstandigheid. Hierdie diëte kan ook ’n inflammatoriese reaksie veroorsaak, wat lei tot miokardialeverstywing met onderliggende hartfibrose. Onlangs is die idee voorgestel dat lisiel- oksidase (LOXL2), 'n ensiem nodig vir die vorming van kollageen en elastienkruisbindingsin die miokardium, 'n belangrike rol speel in hartfibrose en meganiese disfunksie tydens verhoogde hartspanning. Ten spyte van die noodsaaklike rol van LOXL2 as 'n vroeë merker van hartfibrose, is daar 'n tekort aan data oor die regulering van LOXL2 en die rol daarvan in KVS. Verder, in die soektog na nuwe terapeutiese teikens, het hierdie studie gemik om die verbeteringseffekte van AfriplexGRTTM op hoë glukose en palmitaat (HG+PAL)-geïnduseerde stres in 'n in vitro H9c2 selkultuurmodel, en in vivo in ‘n dieet-geïnduseerde Wistar rotmodel van hartfibrose te ondersoek. Addisioneel, is daar 'n definitiewe behoefte om LOXL2-regulering te ondersoek deur middle van epigenetiese meganismes. Dit sal beslis help om 'n manier te vind, ten einde LOXL2 te beheer en te manipuleer, veral in mense, en om sodoende nuwe terapeutiese middele te ontwikkel om hierdie wêreldwye KVS-probleem te beveg. Hierdie studie het dus gepoog om insig te verkry in (1) die meganismes waardeur LOXL2 en stroomafwaartse effektors gereguleer word in fisiologiese toestande wat verantwoordelik is vir KVS-ontwikkeling, (2) of LOXL2-uitdrukking by manlike en vroulike rotte gereguleer word deur DNA- metielasie, en (3) as 'n farmaseutiese kwaliteit van groen rooibos (AfriplexGRTTM) veranderde geenuitdrukking kan verhoed. Metodes: In die in vitro-model, is H9c2-selle vir 24 uur lmet HG+PAL behandel, gevolg deur ‘n verdere 6 uur- behandeling met AfriplexGRTTM, of die bioaktiewemiddel, Aspalatien. Die effek van stresinduksie was geondersoek deur middel van die assessering van ATP and ROS produksie, mitokondriale bioenergie faktore, mitokondriale membraanpotensiaal en apoptose. Selle was ook geoes vir mRNA-uitdrukkingsanalises. Vir die in vivo, dieet-geïnduseerde fibrose model, is Wistar-rotte van manlike en vroulike geslag verskillende obesogeniese diëte gevoer, insluitend 'n hoë vet, hoë suiker (HFHS) dieet, ‘n obesogeniese 1 (OB1) dieet, of 'n obesogeniese 2 (OB2) dieet, oor 'n vasgestelde periode, met of sonder AfriplexGRTTM. liggaamsgewig, voedselinname en bloedglukose was gedurende die studietydperk gemonitor, waarna bloed getrek is vir serumanalise van bloedvette, trigliseriede, insulienvlakke sowel as LOXL2-vlakke. Hartweefsel was ook vesamel vir DNA-metielasie profiele (slegs HFHS-dieet), RNA en proteïenvlak-analises. Resultate: Resultate van die in vitro-studie het gewys dat die HG+PAL-behandeling gelei het tot inflammasie, verhoogde oksidatiewestres en apoptose, sowel as verlaagde mitokondriale membraanpotensiaal. Dit kon egter nie 'n verandering teweegbring in geenuitdrukkingsvlakke van verskillende fibrosemerkers nie, insluitende LOXL2. AfriplexGRTTM- en Aspalatien-behandelings kon nie die nadelige effekte wat deur HG + PAL-behandeling veroorsaak was, teenwerk nie. Resultate van die in vivo studies het aangedui dat die obesogeniese diëte wel die liggaamsgewigte van die diere verhoog het, sowel as die seruminsulien, trigliseried en serum LOXL2, maar geen verskille is waargeneem op 'n geenuitdrukking- of DNA-metileringvlak nie. Verhoogde proteïenuitdrukkingsvlakke het ooreengestem met die verhoogde serumvlakke van LOXL2 in manlike Wistar- rotte. Daarom het ons gepostuleer dat vroeë hartfibrose verwante prosesse moontlik geaktiveer was, wat verdere eksperimente en ondersoeke sal vereis. Gevolgtrekking: Hierdie studie het getoon dat 'n obesogeniese dieet geslagspesifieke veranderinge in metaboliese prosesse kan veroorsaak. Hierdie resultate het ook aangedui dat LOXL2-regulering moontlik aangedryf word deur 'n meganisme wat onafhanklik is van DNA-metielasie en dat AfriplexGRTTM nie LOXL2-geïnduseerde fibrose of hartversteurings in die dieet-geïnduseerde Wistar rotmodel kon verbeter nie. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.format.extent | xxi, 225 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/123588 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Lysyl oxidase-like 2 (LOXL2) | en_ZA |
| dc.subject | Fibrosis | en_ZA |
| dc.subject | DNA methylation | en_ZA |
| dc.subject | Cardiac dysfunction | en_ZA |
| dc.subject | Cardiovascular diseas | en_ZA |
| dc.subject | Diet-induced obesity | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Investigating aberrant DNA methylation patterns in LOXL2 as a potential predictor of diabetes-induced cardiac dysfunction | en_ZA |
| dc.type | Thesis | en_ZA |