The role of Phosphodiesterase 4 in insulin- and phytocannabinoids-induced cardio-protection, and B-adrenergic cardiac damage.

Smith, Angelique (2015-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction: Cardiovascular disease (CVD) is one of the main non-communicable diseases globally contributing to deaths each year. Ischemic heart disease leading to myocardial infarction (or heart attack as it is commonly known) is one of the leading causes of death in the United States (Raven et al. 2008) and in South Africa (Stats SA, 2013). Much research is and has been done to understand the underlying factors that lead to this disease, but this study evaluates the role of phosphodiesterase 4 (PDE 4) in mediating damage or protection during an ischemic event. PDEs function in a coordinated manner under the instruction of various stimuli, mostly known to be from G-protein coupled receptors (GPCRs), in order to promote stimulus-specific cellular physiological effects. PDE4 was considered in this thesis because PDE4 exerts a dominant role in global cellular cAMP hydrolysis in rat cardiomyocytes (Johnson et al 2012) and is responsible for mediating 1- and 2-AR specific effects in normoxic cardiomyocytes (Mika et al 2014, Gregg et al 2010). This is through cAMP compartmentation (Nikolaev et al 2006), which might also be true for ischemia/reperfusion, but has never been evaluated. Thus the PDEs expressed in cardiomyocytes might be responsible for the sensitization of cardiomyocytes to ischemic damage. Aim: The aim was to find an alternative way to intervene during acute myocardial ischemia/infarction, through the GPCR pathways by evaluating the role of phosphodiesterase 4 (PDE 4) in mediating damage or protection during an ischemic event. Methods: Cardiomyocytes were isolated from adult male Wistar rat hearts and cultured overnight on 100ug/ml laminin / entactin (L / E). Following overnight cultures at 5%CO2 and 37°C, experiments were done in modified medium X culture buffer (MMXCB) with different treatment conditions of rolipram (a PDE4 inhibitor), dobutamine (b1AR-agonist), formoterol (b2AR-agonist), isoproterenol (b1- and b2AR-agonist), HU210 (synthetic cannabinoid) and THC (delta-9 tetrahydrocannabinol) to compare adult rat cardiomyocytes (ARCM) attachment, contracture and viability. Cardiomyocytes cultured on L/E were subjected to 20 minutes of simulated ischemia [using MMXCB that contained 3mM 2-deoxyglucose (2DG) and 10mM Sodium dithionite (SDT)] with the different treatment drugs administered during ischemia, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view that was captured using fluorescence microscopy. The cells were analysed according to cell length, morphology and fluorescence intensity with the help of ImageJ Fiji (open source software at http://fiji.sc/Fiji). Results: A Significant difference in the cell length parameter was observed between the normoxic untreated groups compared to the ischemic untreated groups and this indicated that the ischemic conditions were sufficient to result in hypercontracture induced by ischemia/reperfusion. Regardless of eliciting a proper ischemic insult, no treatments administered during ischemia could alter hyper contracture in the cardiomyocytes. Mitochondrial viability, measured in red over green fluorescence intensity did not always portray a significant difference between the normoxic untreated and ischemic untreated groups. The mitochondrial viability did however increase significantly when 10nM HU210 was administered during ischemia compared to ischemia untreated group. 10uM THC increase the viability when compared to ischemia untreated. With the following experiments the results could not be obtained. The rod-shaped ARCMs attachment as measured by percentage cell viability according to morphology, did show a significant decrease after ischemic stimulation, but no differences could be monitored when the different treatment conditions was compared as the percentage viability decreased greatly to levels of 20% and less. Conclusion: This study suggests that B-AR pathways did not have an effect on cardiomyocyte hyper contracture during an severe ischemia insult, nor insulin. It did show that the cannabinoid-receptors increased mitochondrial viability during ischemia and does indeed play a role during ischemia/reperfusion. However, the exact mechanisms for these effects seen are unknown and need further investigation. PDE4 inhibition with and without different agonists also failed to alter hypercontracture. This lack of change in cardiomyocyte survival parameters in response to the different protective and damaging drugs is considered to be a result of the harsh ischemic conditions used. It is recommended to instead incorporate hypoxia with these treatments to evaluate the effects of PDE4 in the presence and absence of b-AR, cannabinoid and insulin signalling.

AFRIKAANSE OPSOMMING: Inleiding: Kardiovaskulêre siekte is een van die grootste nie-oordraagbare siektes wat wêreldwyd bydra tot talle sterftes elke jaar. Iskemiese hartsiekte wat tot miokardiale infarksie (hartaanval, soos dit algemeen bekend staan) lei, is een van die grootste oorsake van sterftegevalle in die Verenigde State van Amerika (Raven et al. 2008) asook in Suid-Afrika (Stats SA, 2013). Baie navorsing is reeds en word tans gedoen om die onderliggende faktore wat tot hierdie siekte lei te verstaan. Hierdie studie evalueer die rol van die ensiem, fosfodiesterase 4 (PDE4) in miokardiale iskemie. Fosfodiesterases funksioneer in 'n gekoördineerde wyse in opdrag van verskeie stimuli, via G-protein gekoppelde reseptore (GPCRs), om sodoende stimulus-spesifieke sellulêre fisiologiese effekte teweeg te bring. Daar is op PDE-4 in hierdie tesis gefokus, vanweë sy dominante rol in die globale sellulêre cAMP hidrolise in rot kardiomiosiete (Johnson et al 2012). Dit is verantwoordelik vir die bemiddeling van b1- en b2-adrenergiese reseptor (AR) spesifieke effekte in normoksies kardiomiosiete (Mika et al 2014, Gregg et al 2010), deur middel van sikliese nukleotied (cAMP) kompartementalisering (Nikolaev et al 2006), wat moontlik ook vir iskemie / reperfusie geld, maar nog nie getoets is nie. Dit is dus moontlik dat die PDEs wat in kardiomiosiete voorkom, verantwoordelik is vir die sensitisering van kardiomiosiete tydens iskemie. Doel: Om 'n alternatiewe manier te vind, deur middel van G-proteïen gekoppelde reseptore (GPCR’s), te om die rol van PDE 4 in miokardiale beskadiging of beskerming tydens iskemie te evalueer. Metodes: kardiomiosiete is uit volwasse manlike Wistar rotharte geïsoleer en oornag op 100ug / ml laminin / Entactin (L / E) gekweek. Na oornag inkubasie met 5% CO2 by 37 °C, is die selle in gemodifiseerde medium X kultuur buffer (MMXCB) blootgestel aan behandeling met verskillende middles soos rolipram, dobutamien, formoterol, isoproterenol, HU210 en THC en die effekte op volwasse rot kardiomiosiet (VRKM) vashegting, kontraktuur en lewensvatbaarheid vergelyk. Die kardiomiosiete gekweek op L / E, is aan 20 minute van gesimuleerde iskemie (MMXCB plus 3mM 2DG en 10mm SDT) blootgestel in teenwoordigheid van bogenoemde middels, met die verskillende behandeling dwelms toegedien tydens iskemie, gevolg deur 15 minute reperfusie. Die lewensvatbaarheid van selle is bepaal deur kleuring met JC-1 en fluoressensie mikroskopie. Die selle is ontleed volgens lengte, morfologie en fluoressensie intensiteit met die hulp van Image J Fiji (Ope toegang tot sagteware deur http://fiji.sc/Fiji). Resultate:'n Beduidende verskil in sellengte is gevind tussen die normoksiese onbehandelde groepe vergeleke met die iskemiese onbehandelde groepe wat toon dat die iskemiese toestande genoeg was om tot behoorlike beskadiging te lei, soos angedui deur die kontraktuur van die selle. Ongeag hierdie beskadiging, het geeneen van die ander middels toegedien tydens iskemie, enige effek op sellengte gehad nie. Mitokondriale lewensvatbaarheid, is gemeet in rooi oor groen fluoressensie intensiteit en het nie altyd 'n beduidende verskil tussen die normoksiese onbehandelde en iskemiese onbehandelde groepe getoon nie. Die mitokondriale lewensvatbaarheid het egter aansienlik verhoog tydens toediening van 10nM HU210 tydens iskemie in vergelyking in vergelyking met die iskemie onbehandelde groep. Dieselfde is gevind toe isoproterenol in kombinasie met rolipram tydens iskemie toegedien is. Daarenteen het 10uM THC die mitokondriale lewensvatbaarheid grootliks verminder in vergelyking met onbehandelde iskemie. Laasgenoemde eksperiment kon egter nie by herhaling dieselfde resultate lewer nie. Die staafvormige VRKMs vashegting is gemeet deur lewensvatbaarheid persentasie van sel soos aangedui deur morfologie, en het 'n beduidende afname na blootstelling aan iskemie getoon. Geen verskille is egter waargeneem tydens blootstelling aan die verskillende middels nie. Gevolgtrekking: Hierdie studie dui daarop dat die beta-adrenergiese paaie nie 'n effek op kardiomiosiete hiperkontraksie tydens 'n iskemiese insident het nie. Dit het wel getoon dat kannaboïed-reseptor stimulasie, ‘n toename in mitokondriale lewensvatbaarheid tydens iskemie teweegbring en moontlik 'n rol tydens iskemie / reperfusie speel. Die presiese meganismes vir die effekte wat waargeneem was, is egter nog onbekend en benodig verdere ondersoek. PDE-4 inhibisie met en sonder verskillende agoniste het ook geen effek op kontraktuur gehad nie. Die gebrek aan verandering in kardiomiosiet oorlewingparameters in hul reaksie op die verskillende beskermende en skadelike middels, kan moontlik aan die erge iskemie waaraan die selle blootgestel is, toegeskryf word. Dit word aanbeveel om die selle aan hipoksie (liewer as iskemie) bloot te stel ten einde die effek van die middels te herevalueer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98618
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