Identification of novel DNA methylation signatures in the development of cardiovascular disease

Date
2021
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Abstract
ENGLISH ABSTRACT: Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, claiming approximately 17.9 million lives annually. The last few decades have seen an exponential increase in the prevalence of major CVD risk factors, such as obesity, insulin resistance (IR), and type 2 diabetes mellitus (T2DM) in underdeveloped countries, including South Africa. This increase is concomitant to escalating CVD incidence and in part due to increased consumption of diets high in fat and sugar, and sedentary lifestyles. Several mechanisms have been implicated in the pathogenesis of diabetic-induced CVD and recently, increasing evidence suggests that dysregulation of the epigenome may play an important role in the development of T2DM and related cardiac complications. More specifically, aberrant DNA methylation has been extensively investigated and implicated in the development of chronic diseases such as obesity, T2DM and CVD. Nonetheless, it has been suggested that some active dietary compounds may reverse this epigenetic phenomenon which allows for the modification of the transcription of critical genes associated with the progression of CVD. Several plant-polyphenols have been reported to influence gene transcription by altering DNA methylation status. Thus, targeting DNA methylation could provide a promising approach for alternative prevention strategies. This study explored the gene expression networks activated during diet-induced CVD and the ability of a green rooibos extract, Afriplex GRT, to alter this consequence. In addition, the study aimed to evaluate aberrant DNA methylation associated with diet-induced CVD to further elucidate pathophysiology. To this end, cardiomyocytes exposed to high glucose and palmitate (HG + Pal) displayed a diminished antioxidant defence system, mitochondrial dysfunction and increased apoptosis, indicative of cardiac stress. Additionally, the combinatory treatment with HG + Pal induced transcriptional changes associated with inflammation, oxidative stress, altered lipid metabolism and increased contractile dysfunction, ultimately promoting the development of atherosclerosis and hypertrophic cardiomyopathy. Interestingly, post treatment with Afriplex GRT or Aspalathin had no significant effect on the metabolic and molecular derangements induced under HG + Pal stress. Similarly, RNA sequencing conducted on cardiac tissue of Wistar rats that received a high fat, high sugar (HFHS) diet, revealed the downregulation of differentially expressed genes (DEGs) involved in host antioxidant activity and inflammatory response, accompanied with an increase in hypertrophic gene expression possibly affecting cardiac muscle functionality. Supplementation with Afriplex GRT™ yielded no high confidence results for the amelioration of the transcriptomic signatures resulting from HFHS diet feeding. To profile DNA methylation throughout disease progression, cardiac tissue of male Wistar rats maintained on a HFHS diet were subjected to whole genome bisulfite sequencing (WGBS). The latter revealed aberrant DNA methylation of genes linked to the phagosome, platelet activation, toll-like receptor signalling and diabetic cardiomyopathy. Furthermore, hypomethylation within the intergenic and gene body regions of several differentially methylated genes (DMGs) overlapped with DEGs identified in the RNA sequencing analysis. Collectively these results demonstrate the ability of the HFHS diet to act as a pathological stimulus capable of inducing altered gene expression and DNA methylation associated with a heightened proinflammatory and lipid metabolism response that increased the risk of CVD development.
AFRIKAANSE OPSOMMING: Kardiovaskulêre siektes (KVSs) bly wêreldwyd die grootste oorsaak van sterftes en eis jaarliks ongeveer 17,9 miljoen lewens. Die afgelope paar dekades het 'n eksponensiële toename in die voorkoms van belangrike KVS-risikofaktore, soos vetsug, insulienweerstandigheid (IW) en tipe 2-diabetes mellitus (T2DM) in onderontwikkelde lande, insluitende Suid-Afrika, plaasgevind. Hierdie toename gaan gepaard met die toenemende voorkoms van KVS, en is deels die gevolg van verhoogde verbruik van diëte met baie vet en suiker, en onaktiewe lewenstyle. Verskeie meganismes is in die patogenese van diabetes-geïnduseerde KVS geïmpliseer en onlangs het toenemende bewyse daarop gedui dat wanregulering van die epigenoom 'n belangrike rol in die ontwikkeling van T2DM en verwante hartkomplikasies kan speel. Meer spesifiek is afwykende DNA-metilering breedvoerig ondersoek en in die ontwikkeling van kroniese siektes soos vetsug, T2DM en KVS geïmpliseer. Nietemin word dit voorgestel dat sommige aktiewe dieetverbindings hierdie epigenetiese verskynsel kan omkeer en die transkripsie van kritieke gene, wat verband hou met die progressie van KVS, kan modifiseer. Daar word berig dat verskeie plant-polifenole geentranskripsie beïnvloed deur die status van DNA-metilering te verander. Dus, deur te fokus op DNA-metilering kan dit 'n belowende benadering tot alternatiewe voorkomingstrategieë bied. Hierdie studie het ondersoek ingestel na die gene uitdrukkings netwerke wat geaktiveer word tydens dieet-geïnduseerde KVS en die vermoë van 'n groen rooibos-ekstrak, Afriplex GRT, om hierdie gevolg te verander. Daarbenewens het die studie ten doel gehad om afwykende DNA-metilering geassosieer met dieet-geïnduseerde KVS te evalueer om patofisiologie verder toe te lig. Vir hierdie doel het kardiomiosiete wat aan hoë glukose en palmitaat (HG + Pal) blootgestel was, 'n verminderde antioksidant-verdedigingstelsel, mitochondriale disfunksie en verhoogde apoptose getoon, wat dui op kardiale spanning. Verder het die kombinasiebehandeling met HG + Pal transkripsionele veranderinge veroorsaak wat verband hou met inflammasie, oksidatiewe spanning, veranderde lipiedmetabolisme en verhoogde kontraktiele disfunksie, wat uiteindelik die ontwikkeling van aterosklerose en hipertrofiese kardiomiopatie bevorder. Dit was interessant dat post-behandeling met Afriplex GRT of Aspalatien geen noemenswaardige effek op die metaboliese en molekulêre afwykings wat onder HG + Pal spanning veroorsaak is, gehad het nie. Net so het RNA-volgordebepaling, wat uitgevoer was op hart weefsel van Wistar-rotte wat 'n dieet met 'n hoë vet, hoë suiker (HVHS) ontvang het, die afregulering van differensieel uitgedrukte gene (DUG'e) wat betrokke is by die gasheer-antioksidantaktiwiteit en inflammatoriese reaksie, aan die lig gebring, met 'n gepaardgaande toename in hipertrofiese geenuitdrukking wat moontlik die hartspier funksionaliteit kon beïnvloed. Aanvulling met Afriplex GRT™ het geen hoë vertrouensresultate opgelewer in die verbetering van die transkriptomiese aanwysings as gevolg van HVHS-dieetvoeding nie. Om die DNA-metilering profiel gedurende die hele siekteprogressie te neem is hartweefsel van manlike Wistar-rotte, wat op 'n HVHS dieet gehou was, onderwerp aan 'n volledige genoom-bisulfiet-volgordebepaling. Laasgenoemde het afwykende DNA-metilering van gene aan die lig gebring wat gekoppel is aan die fagosoom, bloedplaatjie-aktivering, tol- agtige reseptor seinpaaie en diabetiese kardiomiopatie. Verder het hipometilering in die intergeniese streke en geenliggaam van verskeie differensieel gemetileer gene (DMG’e) oorvleuel met DUG'e wat geïdentifiseer is in RNA-volgordebepaling. Gesamentlik toon hierdie resultate die vermoë van die HVHS-dieet om as 'n patologiese stimulus op te tree wat veranderende gene-uitdrukking en DNA-metilering kan veroorsaak, wat gepaard gaan met verhoogde inflammatoriese reaksies en lipiedmetabolisme wat uiteindelik die risiko vir KVS-ontwikkeling verhoog.
Description
Thesis (PhD)--Stellenbosch University, 2021.
Keywords
Cardiovascular disease, DNA -- methylation, Epigenetics, Next generation sequencing, RNA sequencing, Whole genome bisulfite sequencing
Citation