The role of Phosphodiesterase (PDE3) in heart protection.

Labuschagne, S. W. (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction: During a heart attack the blood supply to the heart is reduced significantly, known as ischemia (Reimer & Jennings, 1991). Energy generating pathways that require oxygen become inhibited in the ischemic tissue. The energy decline leads to an increase in ions (calcium, hydrogen, sodium) inside the ischemic heart cells, leading to tissue damage, which is enhanced by reperfusion (Nakamura et al 1999, Piper et al 2004). Hypercontracture, mitochondrial membrane potential and cell death are the hallmarks of ischemia/reperfusion injury. β-adrenergic receptor (β-AR) activation by sympathetic stimuli increases the intracellular cyclic AMP levels, which activates PKA and enhance cell damage during ischemia. Phosphodieterases (PDEs) have been found to play a role in the sensitivity of cardiomyocytes to anoxia induced cell death (Geisbuhler et al., 2002), and PDE3 is known to play a role in β-AR mediated signalling (Christ et al., 2009). Therefore the involvement of PDE3 was evaluated as a regulator of cell survival in the presence and absence of β-AR stimulation (β1-, β2- and β3-AR, separately and together), and insulin administration. Aims: The main aim of this study is to investigate the effective role of the PDE isoform 3, in the cardioprotection induced by insulin versus damage induced by the β-AR and to identify the possible mechanisms involved. Methods: Cardiomyocytes were isolated from Male Wistar rat hearts and cultured overnight in 96-well plates for simulated ischemia/reperfusion experiments the next day. Twelve experimental groups were tested in all objectives including (1) Control; (2) Control + PDE 3 inhibitor; (3) Insulin; (4) Insulin + PDE 3 inhibitor; (5) Dobutamine; (6) Dobutamine + PDE 3 inhibitor; (7) Formoterol; (8) Formoterol + PDE 3 inhibitor; (9) Isoproterenol; (10) Isoproterenol + PDE 3 inhibitor; (11) BRL-37344; and (12) BRL-37344 + PDE 3 inhibitor. These treatments were applied during 20 minutes simulated ischemia induced by 3mM SDT and 10mM 2DG, followed by 60 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells were captured using fluorescence microscopy. The cells were analysed according morphology and fluorescence intensity. Results: In cardiomyocytes that were subjected to 20 minutes simulated ischemia and 60 minutes reperfusion, 3mIU insulin mediated cardioprotection through decreasing cell hypercontracture and increasing cell viability. β2-AR stimulation through the agonist 10uM formoterol also led to protection regarding the cell parameters. Cardiomyocyte protection during ischemia was also elicited when the PDE3 enzyme were inhibited by 10uM milrinone. β1-AR stimulation with 10uM dobutamine appears to have no significant effect regarding damage or protection, while β3-AR stimulation with 10uM BRL37344 resulted in protection. Conclusion: Cardioprotection was elicited with the inhibition of the PDE3 enzyme, but did not have any significant effect on insulin-mediated cell protection. One of the most remarkable observations made, was the fact that β2-AR stimulation with formoterol and β3-AR stimulation with BRL-37344 the most effective triggers of cardioprotection.

AFRIKAANSE OPSOMMING: Inleiding: Tydens 'n hartaanval word die bloedtoevoer na die hart aansienlik verminder, wat bekend staan as isgemie (Reimer & Jennings, 1991). Energie genererende weë wat suurstof benodig word geïnhibeer in die isgemiese weefsel. Die afname in energie lei tot 'n toename in ione (kalsium, waterstof, natrium) in die isgemiese selle, wat lei tot weefsel skade, wat vererger word deur herperfusie (Nakamura et al 1999, Piper et al 2004). Hiperkontraksie, mitochondriale membraan potensiaal en die verlies van selle deur seldood is kenmerke van isgemie/herperfusie beskadiging. β-adrenerge (β-AR) aktivering deur sintetiese stimuli verhoog die intrasellulêre sikliese AMP vlakke, wat PKA aktiveer en selskade verder verhoog tydens isgemie. Daar is bevind dat fosfodiesterases (PDEs) 'n rol speel in die sensitiwiteit van kardiomiosiete aan anoksie geïnduseerde seldood (Geisbuhler et al., 2002), en dit is bekend dat PDE3 ‘n rol speel in β-AR seintransduksiepaaie (Christ et al., 2009). Gevolglik is die betrokkenheid van PDE3 geëvalueer as 'n reguleerder van seloorlewing in die teenwoordigheid en afwesigheid van β-AR sein stimuli (β1-, β2- en β3-AR, afsonderlik en tesame), en insulien toediening. Doelwitte: Die hoofdoel van hierdie studie is om die effektiewe rol van die PDE isoform 3 te ondersoek, in die miokardiale beskerming gebied deur insulien teenoor skade veroorsaak deur die β-AR, en om die moontlike meganismes wat betrokke is te indentifiseer. Metodes: Kardiomiosiete is geïsoleer vanaf Wistar rotharte en oornag in 96 putjie-plate gekultiveer vir gesimuleerde isgemie/herperfusie eksperimente die volgende dag. Twaalf eksperimentïele groepe is getoets in alle doelwitte, insluitend (1) Kontrole; (2) Kontrole + PDE 3 inhibitor; (3) Insulien; (4) Insulien + PDE 3 inhibitor; (5) Dobutamien; (6) Dobutamien + PDE 3 inhibitor; (7) Formoterol; (8) Formoterol + PDE 3 inhibitor; (9) Isoproterenol; (10) Isoproterenol + PDE 3 inhibitor; (11) BRL-37344; en (12) BRL-37344 + PDE 3 inhibitor. Hierdie behandelings is aangewend tydens 20 minute gesimuleerde isgemie, wat bewerkstellig is deur 3mM STD en 10mM 2DG, gevolg deur 60 minute herperfusie. Seloorlewing is bepaal deur die selle met JC-1 te kleur en afbeeldings van selle is gemaak deur middel van fluoresensie mikroskopie. Die selle is ontleed volgens morfologiese en fluoresserende intensiteit. Resultate: In kardiomiosiete wat blootgestel was aan 20 minute gesimuleerde isgemie en 60 minute herperfusie, het 3mIU insulien miokardiale beskerming bewerkstellig deur sel hiperkontraksie te verminder en seloorlewing te verhoog. β2-AR stimulering deur die agonis 10uM formoterol het ook gelei tot beskerming ten opsigte van die sel parameters. Kardiomiosiet beskerming tydens isgemie was ook teweeg gebring met die inhibering van die PDE3 ensiem deur 10uM milrinone. β1-AR stimulering met 10uM dobutamien het geen beduidende effek rakende skade of beskerming getoon nie, terwyl β3-AR stimulering met 10uM BRL37344 tot beskerming gelei het. Gevolgtrekking: Miokardiale beskerming is bewerkstellig deur die inhibering van die ensiem PDE3, maar dit het nie enige noemenswaardige uitwerking op insulien-bemiddelde sel beskerming gehad nie. Een van die mees merkwaardige waarnemings was die feit dat β2-AR stimulering met formoterol en β3-AR stimulering met BRL-37344 die mees doeltreffende bewerker van miokardiale beskerming was.

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