Optimization of experimental conditions and analysis tools for the study of phosphodiesterase-5 in a model of cultured adult rat cardiomyocytes

Botha, Anél (2017-03)

Thesis (MMed)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Part 1 Introduction: Phosphodiesterases (PDEs) hydrolyse cyclic nucleotides that regulate ischemia-reperfusion injury (IRI) in the heart. Phosphodiesterases-5 (PDE5) inhibition increases cyclic guanosine monophosphate (cGMP) levels and thereby promotes cardioprotection. Cannabidiol is a cannabinoid that can alter cGMP levels and induced protection in whole hearts. Cannabidiol-mediated cardioprotection might be controlled by specific PDEs, possibly PDE5. This study aimed to: Evaluate the role of PDE5 inhibition in IRI. Determine whether PDE5 plays a role in cannabidiol-mediated protection. Methods: Cultured adult rat cardiomyocytes were subjected to 20 minutes ischemia, 60 minutes reperfusion, which included mitochondrial staining to measure mitochondrial function with JC-1, followed by fluorescence microscopy and image analysis. A cardioprotective dose of cannabidiol and time of intervention was sought by administration of cannabidiol (0.001 μM, 1 μM and 100 μM) during ischemia and reperfusion, only ischemia, and only reperfusion, respectively. 10 μM Sildenafil was administered during ischemia only to inhibit PDE5. Results: Ischemia-reperfusion reduced cell viability according to morphology by 79 % and mitochondrial function by 50 %. None of the treatments induced cardioprotection. Conclusion: The lack of cardioprotection from cannabidiol and sildenafil might have been due to (1) the ischemic conditions being too harsh, (2) the analysis program being faulty, or (3) unreliable data from morphology analysis. These three points of concern became the basis for the new objectives investigated in Part 2 of this thesis. Part 2 Introduction: Cell viability and mitochondrial function are parameters normally evaluated in cardiomyocytes, and were also used in this study, but cardioprotection could not be found. This raised concerns about the reliability of the image analysis program (ImageJ), the severity of ischemia, and the reliability of the parameters measured. The method used to determine cell viability was especially questioned, because it relies on the researcher to classify rod cells as viable and round cells as dead, which is thus subjective. Morphometry analysis with length over width (L/W) removes the human aspect, allowing cell viability to be determined by classifying cardiomyocytes with L/W ≥ 1.5 as viable. Length on its own is also a morphometric measurement, but is seldom used. Part 2 of this study aimed to: Compare image analysis of ImageJ with that of CellProfiler. Optimize conditions for ischemia-reperfusion and hypoxia-reperfusion. Compare morphology analysis with morphometry analysis. Methods: The sildenafil experimental images from Part 1 were reanalyzed using CellProfiler and the data compared with that found with ImageJ. Ischemia-reperfusion was induced with less harsh conditions for 1 hour, and compared to hypoxia-reperfusion, using cell viability and mitochondrial function. Cell viability was determined by selecting viable cells by rod shape, compared to L/W ≥ 1.5, and length ≥ 55 μm. The average length for hypercontracted cells in the normoxic population was determined, and found to be consistently below 55 μm. Length ≥ 55 μm was chosen as morphometry selection to identify viable cells. Results: Both ImageJ and CellProfiler provided similar data. Cell viability for L/W ≥ 1.5 and length ≥ 55 μm were similar, but higher than morphology, especially for hypoxia-reperfusion, but not for ischemia-reperfusion. L/W ≥ 1.5 and length ≥ 55 μm found differences between normoxia and hypoxia-reperfusion, unlike morphology. The differences can be explained by morphology selecting fewer cells that are perfectly healthy, while morphometry selects more cells with varying degrees of cell injury. Only for ischemia-reperfusion did all parameters provide similar knockdown. This can be explained by ischemia-reperfusion that induced severe injury and hypoxia-reperfusion that induced less injury. Conclusion: The lack of cardioprotection by PDE5 inhibition and cannabidiol was not due to an image analysis error by the program, but might rather be due to ischemia-reperfusion that was too harsh. Conversely, hypoxia-reperfusion induced injury that was not harsh enough. Morphometry selection is biased and unreliable, and morphometry selection should rather be used to evaluate an injured cardiomyocyte population.

AFRIKAANSE OPSOMMING : Deel 1 Inleiding: Fosfodiesterases (FDEs) hidroliseer sikliese nukleotiedes wat iskemie-herperfusie besering (IRB) in die hart reguleer. Fosfodiesterases-5 (FDE5) inhibisie verhoog sikliese guanosien monofosfaat (sGMF) vlakke en bevorder daardeur kardiobeskerming. Cannabidiol is ‘n cannabinoïde wat sGMF vlakke kan verander en induseer beskerming in heel harte. Cannabidiol-bemiddelde kardiobeskerming word moontlik beheer deur spesifieke FDEs, waarskynlik FDE5. Die doelwitte van hierdie studie was om: Die rol van FDE5 inhibisie in IRB te evalueer. Te bepaal of FDE5 ‘n rol speel in cannabidiol-bemiddelde beskerming. Metodes: Gekultuurde volwasse rotkardiomiosiete was onderwerp aan 20 minute iskemie, 60 minute herperfusie, insluitend mitokondriale kleuring om mitokondriale funksie te meet met JC-1, gevolg deur fluoressensie mikroskopie asook beeldanalise. ‘n Kardiobeskermende dosis van cannabidiol en intervensietyd was bepaal deur die administrasie van cannabidiol (0.001 μM, 1 μM en 100 μM) gedurende iskemie en herperfusie, slegs iskemie, en slegs herperfusie, afsonderlik. 10 μM Sildenafil was geadministreer gedurende iskemie alleenlik om FDE5 te inhibeer. Resultate: Iskemie-herperfusie het sellewensvatbaarheid volgens morfologie met 79 % en mitokondriale funksie met 50 % verminder. Geen van die behandelinge het kardiobeskerming geïnduseer nie. Gevolgtrekking: Die gebrek aan kardiobeskerming van cannabidiol en sildenafil is moonlik as gevolg van (1) die iskemie kondisies was te skadelik, (2) die analise program was foutief, of (3) die data van morfologie analise was onbetroubaar. Hierdie drie punte van kommer het die basis geword vir die nuwe objektiewe geëvalueer in Deel 2 van hierdie tesis. Deel 2 Inleiding: Sellewensvatbaarheid en mitokondriale funksie is die parameters wat normaalweg geëvalueer word in kardiomiosiete. Hierdie studie het ook die parameters gebruik, maar kardiobeskerming kon nie gevind word nie. Dit het onsekerheid veroorsaak oor die betroubaarheid van die beeldanalise program (ImageJ), die intensiteit van iskemie, en die betroubaarheid van die geëvalueerde parameters. Die metode wat gebruik was om sellewensvatbaarheid te bepaal was veral bevraagteken, want dit is afhanklik van die navorser om staaf-vormige selle as lewendig en ronde selle as dood te klassifiseer, en is dus subjektief. Morfometrie analise met lengte oor wydte (L/W) verwyder die menslike aspek, en laat toe dat sellewensvatbaarheid bepaal word deur kardiomiosiete met L/W ≥ 1.5 as lewendig te klassifiseer. Lengte op sy eie is ook ‘n morfometrie meting, maar word selde gebruik. Die doel van Deel 2 van hierdie studie was om: Beeldanalise van ImageJ met die van CellProfiler te vergelyk. Die kondisies vir iskemie-herperfusie en hipoksie-herperfusie te optimaliseer. Morfologie analise met morfometrie analise te vergelyk. Metodes: Die sildenafil eksperimentele beelde van Deel 1 is geherevalueer met CellProfiler en die data is vergelyk met die data gevind met ImageJ. Iskemie-herperfusie was geïnduseer met minder skadelike kondisies vir 1 uur, en was toe vergelyk met hipoksie-herperfusie, asook met die gebruik van sellewensvatbaarheid en mitokondriale funksie. Sellewensvatbaarheid was bepaal deur lewendige selle met staaf vorm te kies, en is toe vergelyk met L/W ≥ 1.5, en lengte ≥ 55 μm. Die gemiddelde lengte vir hiper-gekontrakteerde selle in die normoksiese populasie was bepaal, en is gevind om konstant onder 55 μm te wees. Lengte was gekies as morfometriese seleksie ten einde lewendige selle te identifiseer. Resultate: Beide ImageJ en CellProfiler het dieselfde data gehad. Sellewensvatbaarheid vir L/W ≥ 1.5 en lengte ≥ 55 μm was ooreenstemmend, maar hoër as morfologie, spesifiek vir hipoksie-herperfusie, maar nie vir iskemie-herperfusie nie. L/W ≥ 1.5 en lengte ≥ 55 μm het verskille tussen normoksie en hipoksie-herperfusie gevind, anders as morfologie. Die verskille kan verduidelik word deur morfologie wat minder selle selekteer wat net perfek gesond is, terwyl morfometrie selle selekteer wat verskillende grade van selskade toon. Slegs vir iskemie-herperfusie het alle parameters dieselfde besering getoon. Dit kan verduidelik word deur iskemie-herperfusie wat intense skade induseer en hipoksie-herperfusie wat gevolglik minder skade induseer. Gevolgtrekking: Die tekort aan kardiobeskerming deur FDE5 inhibisie en cannabidiol was nie as gevolg van ‘n beeldanalise fout deur die program nie, maar moontlik eerder weens iskemie-herperfusie wat te skadelik was. Inteenstelling, hipoksie-herperfusie het skade geïnduseer wat nie skadelik genoeg was nie. Morfologie seleksie is bevooroordelend en onbetroubaar, en morfometrie seleksie moet eerder gebruik word om ‘n beseerde kardiomiosiet populasie te evalueer.

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