An investigation into the role of ATM protein in mitochondrial defects associated with cardiovascular pathology resulting from insulin resistance

Blignaut, Marguerite (2019-04)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

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.

Agtergrond: Ataxia-telangiectasia (A-T) is ‘n raar, resessiewe siekte wat in die afwesigheid van Ataxia-Telangiectasia gemuteerde proteïen kinase (ATM) ontwikkel. Hierdie komplekse siekte word gekarakteriseer deur neuronale afwykings, ‘n verhoogde risiko vir ontwikkeling van kanker, verhoogde insidensie van insulien weerstandigheid en Tipe 2 diabetes (T2D) sowel as kardiovaskulêre siektes. Mitokondriale afwykings kan lei tot die ontwikkeling van kardiomiopatie en T2D. Vetsug sowel as insulien weerstandigheid dra by tot mitokondriale afwykings. Vorige studies het aangedui dat ATM by die mitokondrion betrokke is, en dat die afwesigheid daarvan kan lei tot ‘n afname in mitokondriale respirasie, ATP sintese, en strukturele veranderinge. Die doel van hiedie studie was om die die rol van ATM in die hart te bepaal met spesifieke fokus op mitokondriale oksidatiewe fosforilasie en mitofagie as moontlike bydraende faktore tot kardiovaskulêre disfunksie in vetsug. Metodiek: ATM uitdrukking is ondersoek in ‘n H9c2 kardiomioblast sel model met die vetsure, palmitaat en oleaat, alleen of in kombinasie met hoë insulien en hoë glukose. Insulien seintransduksie proteïene is deur middel van Western-klad tegniek ondersoek en glukose opname met [3H]2-deoksieglukose. Lokalisering van ATM in die mitokondriale binnemembraan is bepaal in mitoplaste berei uit hartweefsel van manlike Wistar rotte met behulp van digitonien. Kwaliteit van die mitoplaste is met behulp van transmissie elektron mikroskopie (TEM) gekontroleer, en die posisie van ATM is met super resolusie gestruktureerde beligte mikroskopie (SR-SBM) en Western-klad bepaal. Oksidatiewe fosforilasie is polarografies (Clark-tipe elektrode) bepaal in mitokondriē geïsoleer uit (i) harte van jong, kontrole manlike Wistar rotte, geperfuseer met of die draer (DMSO) of die ATM inhibitor, KU60019; (ii) harte van ouderdoms-gepaarde kontrole rotte of rotte gevoer met ‘n hoë kalorie diëet wat vetsug bevorder (DIO), ex vivo geperfuseer met of DMSO, of KU60019 ± insulien of KU60019 ± klorokien. Oksidatiewe fosforilasie is bepaal met koolhidraat (glutamaat+malaat) of vetsuur (palmitoyl-L-karnitien+malaat) substrate en proteïen uitdrukkingsvlakke van ATM asook merkers van mitofagie en mitokondriale fissie is bepaal met Western-klad. Die redoks status van NAD(P)H is bepaal met 2-foton fluoresensie leeftyd visualiserings mikroskopie in H9c2 selle met of KU60019 óf DMSO. Resultate: Hierdie studie het getoon dat (i) hoë vetsuur vlakke asook insulien die uitdrukking van ATM beïnvloed; ii) ATM op die mitokondriale binnemembraan gelokaliseer is; iii) Inhibisie van ATM onderdruk mitokondriale koolhidraat-geïnduseerde mitokondriale oksidatiewe fosforilasie (p=0.0024), moontlik deur Kompleks I. Hierdie waarneming is gesteun deur die bevinding dat ATM inhibisie NADPH vlakke (p=0.02) laat afneem en NADH vlakke laat toeneem in H9c2 selle. iv) Die effek van ATM onderdrukking op oksidatiewe fosforilasie is onafhanklik van diēet. v) ATP sintese asook die respiratoriese kontrole indeks het verbeter in die teenwoordigheid van insulien(p<0.005). vi) Die onderdrukking van ATM met KU60019 het ook mitokondriale fissie verlaag (p=0.0038). vii) Die afname in autofagie membraan merker, LC3-II (p<0.0001), blyk weens die sitosoliese rol van ATM te wees. Gevolgtrekking: ATM is gelokaliseer op die mitokondriale binnemembraan en die inhibisie daarvan beïnvloed mitokondriale ATP sintese, moontlik deur Kompleks I substraat verbruik. Die inhibsie van ATM het nie ‘n rol gespeel in mitokondriale oksidatiewe fosforilasie in vetsug nie, maar het wel mitofagie ontwrig as ook mitokondriale fissie verlaag.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105997
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