The role of mitophagy in damage incurred by ischaemia/reperfusion

Mzezewa, Sibonginkosi Roselyn (2017-03)

Thesis (MMed)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Background and aims: Mitophagy is a specialised process of autophagy whereby dysfunctional mitochondria are removed. Mitophagy has been increasingly recognised as an essential process to sustain a healthy pool of mitochondria for cardiomyocytes as mitochondria are critical for cardiac function. Mitophagy has also become of interest as a role player in cardiovascular pathology: previous studies showed that autophagy is activated during ischaemia/reperfusion injury, but whether it is beneficial or detrimental is still unsure. The aims of this study were to evaluate the role of mitophagy in myocardial ischaemia/reperfusion and its association with cardio protection, mitochondrial function and the signalling events of mitophagy. Use was made of 3-methyl adenine (3-MA), an inhibitor of autophagy and FCCP, an uncoupler of mitochondrial oxidative phosphorylation and stimulant of mitophagy, in order to manipulate the process of mitophagy. Methods: Hearts from male Wistar rats were perfused ex vivo and subjected to 25 min global ischaemia or ischaemia followed by 20 min reperfusion with or without pre-or post-ischaemic 3-MA (1 mM) administrations well as pre-ischaemic/20 min reperfusion FCCP administration. Mitochondria were isolated (KCl-EDTA) for measurement of oxidative phosphorylation with glutamate, malate and palmitoyl-L-carnitine as substrates using a Clark electrode. TOM70, PINK1, Parkin and P62/SQSTM1, LC3, BNIP3L/Nix and Beclin1 accumulation were the mitophagy and autophagy markers determined by Western blotting. A separate group of hearts were subjected to 35 min regional ischaemia/60 min reperfusion with drugs administered pre-ischaemia. Infarct size was determined by Triphenyltetrazolium chloride staining. Citrate synthase levels as measurement of intact mitochondria, were determined using an assay kit. ATP levels for certain experiments were measured using HPLC. Results Mitochondria isolated from hearts treated with 1 mM 3-MA post ischaemia presented with increased QO2 (S3) in the carbohydrate substrate and increased RCI levels in fatty acid substrate. 100 nM FCCP also resulted in increased mitochondrial respiration as indicated by increased RCI and increased QO2(S3) respiration in the fatty acid substrate, but was associated with increased mitochondrial uncoupling indicated by lower ADP/O, and hence decreased oxidative phosphorylation for both states 3 and 4. 250 nM FCCP led to complete mitochondrial uncoupling. Neither drug affected citrate synthase activity. LC3A/B-II/I ratios were lower in both ischaemic and reperfused hearts in comparison to baseline, while 3-MA had no effect on these levels. BNIP3L/Nix levels were upregulated in reperfusion versus in ischaemia, whereas the opposite effect was observed for Parkin and P62/SQSTM1 levels. 3-MA post-ischaemic treatment resulted in increased PINK1. 100 nM FCCP yielded increased TOM70 in comparison to 250 nM but the opposite was observed with Parkin levels. 3-MA had no effect on infarct size whereas both 100 nM and 250 nM FCCP reduced infract size (p=0.0001). Both drugs did not affect functional recovery, however, 250 nM FCCP led to complete mechanical heart failure and was associated with significantly higher levels of unconverted ADP. Conclusion: Manipulation of mitophagy affects the outcome of ischaemia/reperfusion: Downregulation by 3-MA was (i) of little effect to hindering mitophagy in certain protocols indicated by increased mitochondrial PINK1 levels, however (ii) it yielded increased mitochondrial respiration, but (iii) rendered poor heart functional recovery and (iv) had no effect on the infarct size. FCCP (100 nM) upregulation of mitophagy, was (i) associated with cardio protection with (ii) decreased infarct size, but (iii) uncoupling effects of FCCP led to decreased oxidative phosphorylation rates and hence (iv) decreased cellular levels of ATP resulting in (v) poor functional recovery observed.

AFRIKAANSE OPSOMMING : Agtergrond en doelwitte: Mitofagie is ‘n gespesialiseerde vorm van outofagie waardeur disfunksionele mitokondrieë verwyder word. Mitofagie word tans beskou as ‘n essensiële proses om te verseker dat die poel van beskikbare mitokondrieë in ‘n kardiomiosiet goed funksioneer, aangesien hulle krities is vir kardiale funksie. Mitofagie het ook belangstelling ontlok weens die moontlike rol daarvan in kardiovaskulêre patologie: studies het aangetoon dat outofagie gedurende iskemie/herperfusie besering geaktiveer, die voor- of nadele daarvan is egter onseker. Die doelwitte van hierdie studie was om die rol van mitofagie gedurende miokardiale iskemie/herperfusie te evalueer en die verband daarvan met beskerming, mitokondriale funksie en die seintransduksie geassosieer met mitofagie, te ondersoek. Om hierdie prosesse te manipuleer, is gebruik gemaak van 3-metiel adenien (3-MA) as inhibitor van outofagie en FCCP, ‘n ontkoppelaar van oksidatiewe fosforilasie en dus stimulant van mitofagie. Metodes: Harte van manlike Wistar rotte is ex vivo perfuseer en onderwerp aan 25 min globale iskemie gevolg deur 20 min herperfusie met of sonder pre- of postiskemiese toediening van 3-MA (1 mM) sowel as pre-iskemiese toediening van FCCP. Mitokondrieë is geïsoleer (KCl-EDTA) vir bepaling van die oksidatiewe fosforileringspotensiaal met glutamaat, malaat en palmitoïel-L-karnitien as onderskeie substrate, deur ‘n Clark-tipe elektrode te gebruik. TOM70, PINK1, Parkin en P62/SQSTM1, LC3, BNIP3L/Nix en Beklin1 akkumulering as merkers van mitofagie en outofagie, is deur middel van Westerse kladtegnieke bepaal. ‘n Verdere groep harte is onderwerp aan 35 min streeksiskemie/60 min herperfusie met middels voor iskemie toegedien, vir die bepaling van infarktgrootte deur middel van trifenieltetrazolium chloriedkleuring. Sitraatsintasevlakke is deur middel van ‘n kommersiële essai bepaal as maatstaf van die intakte, respirerende mitokondrieë per mg proteien. Mitokontriale ATP produksie is in sekere eksperimente deur middel van HPLC analise bepaal. Resultate: Mitokondrieë berei uit harte wat met 1mM 3-MA na iskemie behandel is, het ‘n verhoogde QO2(3) met glutamaat/malaat en in verhoogde RKI met palmitoïel-L-karnitien/malaat as substrate getoon. 100 nM FCCP het mitokondriale respirasie verhoog soos aangedui deur ‘n verhoogde RKI en QO2(S3) respirasie met palmitoïel-karnitien/malaat as substrate, maar het verhoogde mitokondriale ontkoppeling, aangedui deur ‘n laer ADP/O verhouding, dus verlaagde oksidatiewe fosforilering snelheid vir beide staat 3 en 4, tot gevolg gehad. 250 nM FCCP het mitokondrieë volledig ontkoppel. Nie een van die middels het die sitraatsintase aktiwiteit beïnvloed nie. Die LC3A/B-II/I verhouding was laer in beide iskemie en herperfuseerde harte in vergelyking met die basislyn terwyl 3-MA geen effek hierop getoon het nie. BNIP3L/Nix vlakke was verhoog in herperfusie vs iskemie terwyl die teenoorgestelde effek met Parkin en P62/SQSTM1 vlakke waargeneem is. Post-iskemiese behandeling met 3-MA het verhoogde PINK1 tot gevolg gehad. 100 nM FCCP het TOM70 uitdrukking verhoog in vergelyking met 250 nM maar die teenoorgestelde is waargeneem vir Parkin vlakke. 3-MA het geen effek op infarktgrootte gehad nie terwyl beide 100 nM en 250 nM infarktgrootte verklein het (p=0.0001). Beide middels het geen effek op funksionele herstel gehad nie maar 250 nM FCCP het totale meganiese hartversaking veroorsaak en ook gelei tot beduidende hoër vlakke van oorblywende ADP. Gevolgtrekking: Manipulering van mitofagie het die uitkoms van iskemie/herperfusie beïnvloed: afregulering met 3-MA het (i) baie min effek op mitofagie gedurende sekere protokolle gehad, aangedui deur die verhoogde PINK1 vlakke, alhoewel (ii) dit mitokondriale respirasie verhoog het maar (iii) tot swak funksionele herstel gelei het en (iv) geen effek of infarktgrootte gehad het nie. Opregulering van mitofagie met 100 nM FCCP was (i) geassosieer met miokardiale beskerming en (ii) kleiner infarktgrootte, maar (iii) die ontkoppelingseffekte van FCCP het oksidatiewe fosforileringssnelheid en dus (iv) ATP vorming verlaag met gevolglike (v) swak funksionele herstel.

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