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Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases
Thesis (PhD)--Stellenbosch University, 2020.
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Stellenbosch : Stellenbosch University
2020
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| _version_ | 1867613744413016064 |
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| access_status_str | Open Access |
| author | Ndlovu, Zibele |
| author2 | Marais, Erna |
| author_browse | Marais, Erna Ndlovu, Zibele |
| author_facet | Marais, Erna Ndlovu, Zibele |
| author_sort | Ndlovu, Zibele |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2020. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/108101 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:00.939Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/108101 Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases Ndlovu, Zibele Marais, Erna Strijdom, Hans Stellenbosch University. Faculty of Medical Sciences. Dept. of Biomedical Sciences: Medical Physiology. Ischeamia-reperfusion UCTD Obesity Cardiovascular disease Thesis (PhD)--Stellenbosch University, 2020. ENGLISH ABSTRACT: Introduction and aims: The strong association between obesity and cardiovascular diseases (CVD) stresses the necessity of elucidating the underlying molecular mechanisms linking these pathologies. Evidence suggests that Epac (exchange protein directly activated by cAMP) could be a new therapeutic target for obesity and CVD. This study aimed to elucidate the role of Epac in (i) myocardial I/R injury of ex vivo hearts from normal weight; and diet-induced obese rats; (ii) vascular reactivity of ex vivo aortas from diet-induced obese rats and (iii) free fatty acid (FFA)-induced endothelial dysfunction in rat aortic endothelial cells (RAECs). Methods: Male Wistar rats of normal weight (250 to 350 g) or receiving a high-calorie diet (HC) for 16 weeks or age-matched controls (CD), were used. Mechanical function of isolated perfused working rat hearts were evaluated. Regional ischaemia (35 minutes) was followed by 60 minutes reperfusion and infarct size determination. Hearts were perfused with a selective Epac1 agonist (8-CPT-2’-O-Me-cAMP, CPT, 2 μM) or antagonist (ESI-09, 5 μM) for 10 minutes before (pre-treatment) or after (posttreatment) regional ischaemia and also post-treated with MEK-ERK inhibitor (PD98059, 10 μM) or PKB inhibitor (A6730, 2.5 μM). Thoracic aortas were isolated from rats fed either the HC or CD diet. Aortas were pre-incubated for 15 min with CPT, ESI-09 or nitric oxide synthase (NOS) inhibitor (L-NAME, 100 μM). Vascular reactivity was evaluated by phenylephrine-precontraction followed by acetylcholine-induced relaxation. Cardiac and aortic signalling proteins were detected with Western blotting. Cultured RAECs were supplemented with FFAs (Palmitic acid and Oleic acid) to induce endothelial injury. The effects of FFAs, CPT and ESI-09 on intracellular levels of NO, superoxide (ROS) and cell viability were determined with fluorescence-based assays. Results: After 16 weeks the HC animals presented with significantly increased body weight and visceral fat. In hearts, HC diet had no deleterious effects on post-ischaemic cardiac function and infarct size. Post-treatment with CPT (2 μM) reduced infarct size and improved cardiac recovery. This protection was abolished by post-treatment with A6730 in HC hearts, but not with PD98059. Furthermore, CPT increased CaMKII activation but reduced eNOS phosphorylation. Epac inhibitor (ESI-09, 5 μM) was detrimental to cardiac function. This was associated with: decreased phosphorylated ERK1/2 and GSK-3b; increased phosphorylated AMPK, ULK-1 and PARP cleavage. HC diet did not adversely affect vascular reactivity. CPT significantly improved vasorelaxation in both diets which were abolished by ESI-09. CPT induced vasorelaxation in a dose-dependent manner in both diets via NOS. ESI-09 reduced PKB phosphorylation in CD aortas and increased AMPK phosphorylation in both diets. In normal RAECs CPT improved NO production and reduced intracellular ROS. CPT could not reverse the detrimental effect of FFAs on RAECs. Conclusion: Post-ischaemic Epac activation is cardioprotective against I/R injury in ex vivo isolated rat hearts from HC, partially via the PKB pathway. Epac activation improved vasorelaxation via NOS in ex vivo aortas from both diets. In addition, Epac activation increased NO production and reduced ROS generation in normal RAECs. Therefore Epac may be a potential therapeutic target in the protection against CVD. AFRIKAANSE OPSOMMING: Inleiding en doelstellings: Die sterk verband tussen vetsug en kardiovaskulêre siektes (KVS) beklemtoon die noodsaaklikheid om die onderliggende molekulêre meganismes wat hierdie patologieë verbind, te verklaar. Bewyse dui daarop dat Epac (‘n uitruil-proteïen wat direk deur cAMP geaktiveer word) 'n nuwe terapeutiese teiken vir vetsug en KVS kan wees. Hierdie studie het ten doel gehad om die rol van Epac in (i) miokardiale isgemie-herperfusie (I/R)-besering van ex vivo-harte van óf normale gewig óf dieet-geïnduseerde vetsugtige rotte; (ii) vaskulêre reaktiwiteit van ex vivo aortas van dieet-geïnduseerde vetsugtige rotte en (iii) vrye vetsuur (VVS) geïnduseerde endoteel disfunksie in rot aorta endoteelselle (RAEC), te ondersoek. Metodes: Manlike Wistar-rotte van 'n normale gewig (250 tot 350 g) of 'n hoë-kaloriedieet (HC) vir 16 weke of 'n ouderdomskontrole (CD), is gebruik. Meganiese funksie van geïsoleerde geperfuseerde werkende rot harte is geëvalueer. Streeks-isgemie (35 minute) is gevolg deur herperfusie (60 minute) en infarkt-grootte bepaling. Harte is behandel met 'n selektiewe Epac1-agonis (8-CPT-2'-O-Me-cAMP, CPT, 2 μM) of 'n antagonis (ESI-09, 5 μM) vir 10 minute voor (pre-behandeling) of na (postbehandeling) streeks-isgemie en ook post-behandeling met MEK-ERK-inhibitor (PD98059, 10 μM) of PKB-inhibitor (A6730, 2.5 μM). Torakale aortas is geïsoleer van rotte wat óf die HC- óf CD-dieet gevoer is. Aortas is 15 minute vooraf geïnkubeer met CPT, ESI-09 of stikstofoksied sintase (NOS) inhibitor (L-NAME, 100 μM). Vaskulêre reaktiwiteit is geëvalueer deur fenielefrien-prekontraksie gevolg deur asetielkoliengeïnduseerde ontspanning. Met Western blot analise is hart- en aorta-seineproteïene bepaal. Gekultuurde RAEC is gesupplementeer met VVSe (Palmitaat en Oleaat) om endoteelbesering te veroorsaak. Die effekte van VVSe, CPT en ESI-09 op intrasellulêre vlakke van NO, superoksied (ROS) en sel-oorlewing is bepaal met behulp van fluoressensie-gebaseerde toetse. Resultate: Na 16 weke het die HC-diere se liggaamsgewig en viserale vet aansienlik vermeerder. In die harte het HC-dieet geen nadelige gevolge gehad op postisgemiese hartfunksie en infarkt-grootte nie. Post-behandeling met CPT (2 μM) het infarkt-grootte verminder en die funksionele herstel verbeter. Hierdie beskerming is opgehef deur post-behandeling met A6730 in HC-harte, maar nie met PD98059 nie. Verder het CPT die aktivering van CaMKII verhoog, maar eNOS-fosforilering verminder. Epac-antagonis (ESI-09, 5 μM) was nadelig vir die hartfunksie. Dit was geassosieer met: verminderde gefosforileerde ERK1/2 en GSK-3b; verhoogde gefosforileerde AMPK, ULK-1 en PARP splitsing. HC-dieet het nie die vaskulêre reaktiwiteit benadeel nie. CPT het in beide diëte vaso-verslapping aansienlik verbeter, wat deur ESI-09 opgehef is. CPT het vaso-verslapping op 'n dosis-afhanklike manier in beide diëte via NOS, veroorsaak. ESI-09 verminder PKB-fosforilering in CD-aortas en verhoog AMPK-fosforilering in beide diëte. In normale RAEC's het CPT die NO produksie verbeter en die intrasellulêre ROS verminder. CPT kon nie die nadelige uitwerking van VVSe op RAEC's omkeer nie. Gevolgtrekking: Post-iskemiese Epac-aktivering is kardiobeskermend teen I/Rbeserings in ex vivo geïsoleerde rotteharte van HC, gedeeltelik via die PKB-pad. Epacaktivering het vaso-verslapping, via NOS in ex vivo aortas van beide diëte, verbeter. Boonop het Epac-aktivering NO produksie verhoog en ROS-ontwikkeling in RAEC's verminder. Daarom kan Epac 'n potensiële terapeutiese teiken in die beskerming teen KVS wees. Doctoral 2020-02-25T17:37:03Z 2020-04-28T12:19:14Z 2020-02-25T17:37:03Z 2020-04-28T12:19:14Z 2020-03 Thesis http://hdl.handle.net/10019.1/108101 en_ZA Stellenbosch University 275 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Ischeamia-reperfusion UCTD Obesity Cardiovascular disease Ndlovu, Zibele Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases |
| title | Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases |
| title_full | Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases |
| title_fullStr | Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases |
| title_full_unstemmed | Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases |
| title_short | Epacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases |
| title_sort | epacs exchange protein directly activated by camp role in obesity induced cardiovascular diseases |
| topic | Ischeamia-reperfusion UCTD Obesity Cardiovascular disease |
| url | http://hdl.handle.net/10019.1/108101 |
| work_keys_str_mv | AT ndlovuzibele epacsexchangeproteindirectlyactivatedbycamproleinobesityinducedcardiovasculardiseases |