Exploring features of oxidative stress and senescence in the H9c2 cardiomyoblast cell line

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dc.contributor.advisorHuisamen, Barbaraen_ZA
dc.contributor.advisorSadie-Van Gijsen, Hanélen_ZA
dc.contributor.authorHarries, Sarahen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.en_ZA
dc.date.accessioned2023-03-01T10:57:43Zen_ZA
dc.date.accessioned2023-11-16T08:28:02Zen_ZA
dc.date.available2023-03-01T10:57:43Zen_ZA
dc.date.available2023-11-16T08:28:02Zen_ZA
dc.date.issued2023-03en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH ABSTRACT: Background: Age-related cardiovascular disease is one of the largest causes of mortality globally. Senescent cells in the heart have emerged as a possible contributor to cardiac disease progression; however, studies elucidating the mechanisms explaining this phenomenon are scarce. The protein kinase Ataxia Telangiectasia Mutated (ATM) has been shown to be involved in DNA damage repair, cellular redox homeostasis, and mitochondrial function, and therefore may play a role in regulating senescence in heart cells. Cardiomyocytes are the most common cell-type in the heart; however, due to logistical and ethical considerations, cardiomyocytes are not always a feasible model for cardiac cell research. Therefore, the aim of this study was to determine if the H9c2 cardiomyoblast cell-line can exhibit features of induced senescence as a foundation to study the role of ATM in cardiac cell senescence. Methods: Early and late passage H9c2 cardiomyoblast cells (EP and LP, respectively) were compared in terms of growth rate, secretome and responsiveness to exogenous hydrogen peroxide and retinoic acid, and were then assessed for markers of senescence and changes in oxidative stress status. Markers measured included intracellular reactive oxygen species (ROS), senescence-associated betagalactosidase (SA-βgal) staining, gene expression levels of p16, p21 and ATM, and protein levels of total and phosphorylated ATM, p53 and H2AX. The anti-senescent properties of the antioxidant polyphenol resveratrol were also explored in EP and LP H9c2 cells. Results: EP and LP cells exhibited little evidence of oxidative stress and senescence and displayed high proliferative capacity, no increase in intracellular ROS and no increase in senescent markers. However, LP cells exhibited upregulation of the DNA damage marker γH2AX and the DNA repair protein ATM. Unexpectedly, a high concentration (25 µM) of resveratrol had a detrimental effect on EP and LP cell culture density and significantly upregulated p21 gene expression in EP cells. Discussion: Despite an increase in DNA damage markers and DNA repair proteins in LP H9c2 cells, other markers of senescence were absent, suggesting that the cells were able to repair the damage without committing to senescence. Intriguingly, a high concentration of resveratrol was detrimental to both EP and LP cultures, and consequently, the use of resveratrol as a cardioprotective antioxidant should be https://scholar.sun.ac.za iv questioned and may be dose-dependent. In conclusion, the H9c2 cells overall showed a resistance to respond to various damaging stimuli. Therefore, this cardiomyoblast cell line may not be suitable for research within the context of senescence owing to its limited ability to develop features of senescence.en_ZA
dc.description.abstractAFRIKAANS OPSOMMING: Agtergrond: Ouderdoms-verwante kardiovaskulêre siektes is een van die grootste oorsake van sterftes wêreldwyd. Sel-veroudering in die hart blyk by te dra tot kardiovaskulêre siekte-progressie, maar studies wat die meganismes rondom hierdie verskynsel bestudeer is skaars. Die proteïen kinase ATM is bevind om betrokke te wees by die herstel van DNA-skade, sellulêre redoks-balans en mitochondriale funksie, en mag dus ‘n rol speel in die regulering van sel-veroudering in hartselle. Kardiomiosiete is die mees algemene sel-tipe in die hart, maar weens logistieke en etiese oorwegings is kardiomiosiete nie altyd ‘n werkbare model vir hartselnavorsing nie. Die doel van hierdie studie was dus om te bepaal of die H9c2 kardiomioblastsellyn eienskappe van sel-veroudering kan ontwikkel, as ‘n grondslag om die rol van ATM in hartsel-veroudering te kan bestudeer. Metodes: H9c2 selle wat laer en hoër aantal selverdelings ondergaan het (onderskeidelik LS en HS selle) is vergelyk in terme van groeikoers, sekretoom en reaksie op eksogene waterstofperoksied en retinoïensuur, en merkers van selveroudering en oksidatiewe stres is gemeet. Hierdie merkers het ingesluit: intrasellulêre reaktiewe suurstofspesies (RSS), verouderings-geassosieerde betagalaktosidase (β-gal) kleuring, geen-uitdrukking van p16, p21 en ATM, en totale en gefosforileerde proteïenvlakke van ATM, p53 en H2AX. Die anti-verouderingswerking van die anti-oksidant resveratrol is ook ondersoek in LS en HS H9c2 selle. Resultate: LS en HS selle het min bewyse van oksidatiewe stress en sel-veroudering getoon, met hoë selverdelingskapasiteit, geen toename in intrasellulêre RSS en geen toename in verouderingsmerkers nie. ‘n Toename in die DNA skade-merker γH2AX en die DNA herstel-proteïen ATM is wel waargeneem in HS selle. ‘n Hoë konsentrasie van resveratrol (25 µM) het onverwags ‘n skadelike uitwerking op LS en HS selkultuurdigtheid gehad, en het ook geenuitdrukking van p21 in LS selle laat toeneem. Bespreking: Ten spyte van ‘n toename in merkers van DNA skade en herstel in HS selle, was ander merkers van sel-verourdering afwesig in hierdie selle, wat aandui dat die selle wel in staat was om die DNA-skade te herstel sonder om sel-veroudering te ondergaan. Die onverwagse bevinding dat ‘n hoë konsentrasie van resveratrol skadelik was vir beide LS en HS selle dui daarop dat die gebruik van resveratrol as ‘n hart-beskermende anti-oksidant bevraagteken kan word, en dalk dosis-afhanklik kan https://scholar.sun.ac.za vi wees. Ter opsomming, H9c2 selle het in die algemeen geblyk om weerstandig te wees teen ‘n verskeidenheid skadelike insette. Dit dui daarop dat hierdie kardiomioblastsellyn nie gepas is vir navorsing aangaande sel-veroudering nie, as gevolg van beperkte kapasiteit om kenmerke van sel-veroudering te ontwikkel.af_ZA
dc.description.versionMastersen_ZA
dc.embargo.terms2023-09en_ZA
dc.format.extentxix, 113 pages : illustrationsen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/128691en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshCardiovascular diseases in old ageen_ZA
dc.subject.lcshOxidative stressen_ZA
dc.subject.lcshCells -- Mechanical propertiesen_ZA
dc.subject.lcshHeart -- Agingen_ZA
dc.subject.lcshAtaxia telangiectasiaen_ZA
dc.subject.lcshHeart cellsen_ZA
dc.subject.lcshDNA replicationen_ZA
dc.titleExploring features of oxidative stress and senescence in the H9c2 cardiomyoblast cell lineen_ZA
dc.typeThesisen_ZA
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