Local cAMP signalling and phosphodiesterase activity in an in vitro model of cardiac hypertrophy

Fields, Laura Ashley (2013) Local cAMP signalling and phosphodiesterase activity in an in vitro model of cardiac hypertrophy. PhD thesis, University of Glasgow.

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Abstract

Cardiac hypertrophy often develops to compensate for hemodynamic overload and therefore, in its early stages, hypertrophy is considered to be an adaptive response. Nonetheless, prolonged exposure to a hypertrophic stimulus is associated with heart failure. In the heart, the compartmentalisation of the cAMP/ PKA signalling pathway plays a critical role to achieve the specificity of response and maintains regular cardiac function. Alterations in this signalling pathway have been linked to the pathophysiology of cardiac hypertrophy. Phosphodiesterases (PDEs) provide the only means of hydrolysing cAMP and therefore are essential components in the spatial and temporal control of the cAMP response. By restricting the diffusion of cAMP, PDEs prevent unspecific activation of PKA and phosphorylation of downstream targets. PDEs are therefore able to regulate the kinetics of cAMP signalling dynamics. In this study, an in vitro model of chronic catecholamine-induced cardiac hypertrophy of adult rat ventricular myocytes (ARVM) was utilised. This model allowed the investigation of the function of PDEs in regulating compartmentalised cAMP signals in cardiac hypertrophy. Using FRET-based cAMP sensor Epac1_camps fused to the unique dimerisation/docking domain sequences that anchors PKA‐RI and PKA‐RII subunits to AKAPs, this study demonstrated that, similar to neonatal rat ventricular myocytes (NRVM), adult myocytes also display restricted cAMP diffusion. These cAMP microdomains are regulated by different families of PDEs. In particular, PDE2, PDE3 and PDE4 appear to control the pool of cAMP generated in the PKA-RI compartment, whereas only PDE2 and PDE4 were found to modulate cAMP in the PKA-RII compartment in ARVM. In the in vitro cardiac hypertrophy model, a reduction in cAMP generation was detected upon β-adrenergic stimulation and altered PDE activity was visualised using FRET-based imaging. This investigation showed that PDE2 activity is significantly increased in the PKA-RII compartment of hypertrophic cardiac myocytes, while an overall reduction in PDE3 activity was detected. Immunofluorescence experiments revealed altered PDE4 localisation in hypertrophic myocytes. Advances in cyclic nucleotide signalling research, in particular of the activity and regulation of PDEs, have shown that an interaction between the cAMP and cGMP signalling pathways exists. Integration between these two pathways is mediated by the modulation of cAMP‐degrading PDEs by cGMP. Allosteric binding of cGMP to the GAF domains of PDE2 enhances its activity, whereas cGMP reduces the activity of PDE3 by acting as a competitive inhibitor. PDE2 and PDE3 therefore may act as a connection between these two signalling cascades and it is possible to predict the existence of distinct signalling units in vivo in which cGMP, by acting on PDE2 or PDE3, can selectively modulate cAMP levels. Intracellular cGMP generated by stimulation of the particulate GC (pGC) by atrial natriuretic peptide (ANP) or stimulation of the soluble GC (sGC) by the NO donor SNAP is compartmentalised into discrete microdomains. Stimulation of the pGC had no effect on cAMP signalling in the PKA-RII compartment. Activation of sGC generated a pool of cGMP which lead to a reduction in cAMP response in the PKA-RII compartment upon β-AR stimulation. Both GCs generated cGMP in the PKA-RI compartment which lead to an increase in cAMP response. Further investigation revealed that cGMP is able to modulate cAMP signalling in the PKA-RI compartment by PDE3 and by PDE2 in the PKA-RII compartment. It was hypothesised that the observed differences in cAMP signalling and PDE contribution in hypertrophic myocytes in the PKA-RI and PKA-RII subcellular compartments may be due to variations in the pools of cGMP. Employing genetically encoded FRET-based biosensors for cGMP targeted to PKA‐RI and PKA‐RII compartments, basal levels of cGMP were found to be significantly increased in both compartments of hypertrophic myocytes which could explain the altered PDE2 and PDE3 activity in hypertrophic ARVM. Finally, this study shows that PDE2 activity is necessary to achieve full development of the catecholamine-induced hypertrophic response. Pharmacological inhibition of PDE2 with Bay 60-7550 prevents NE-induced cardiomyocyte hypertrophy. Overexpression of PDE2A2 was found to induce hypertrophic growth, even in the absence of increased adrenergic drive, thus indicating that PDE2 activity promotes development of cardiomyocytes hypertrophy. PDE2 localisation is vital for its regulation of hypertrophic growth. Displacement of endogenously active PDE2 from its specific intracellular localisation, using a catalytically inactive PDE2A2, was sufficient to counteract catecholamine-induced hypertrophic growth As PDE2 is a dual specific PDE, it was important to establish whether the anti-hypertrophic effects of PDE2 inhibition were mediated by activation of the cGMP/ PKG or cAMP/ PKA signalling pathways. The data presented here, show that the effects of pharmacological inhibition of PDE2 is PKA dependent. Together, these findings confirm the involvement of PDE2 in the progression of hypertrophy in cardiomyocytes and indentify PDE2, specifically coupled to the PKA-RII compartment, as a possible novel target for the development of therapeutic treatment for hypertrophy.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: cyclic AMP, hypertrophy, phosphodiesterase, compartmentalisation, cardiomyocyte, PDE2
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Funder's Name: UNSPECIFIED
Supervisor's Name: Zaccolo, Prof. M.
Date of Award: 2013
Depositing User: Miss L A Fields
Unique ID: glathesis:2013-4755
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 Jan 2014 08:52
Last Modified: 10 Jan 2014 08:54
URI: http://theses.gla.ac.uk/id/eprint/4755

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