| Summary: | The ubiquitous second messenger molecule, cyclic adenosine monophosphate (cAMP), regulates a multitude of cellular functions, with Protein Kinase (PK)A being the major effector of its actions. There is increasing evidence to suggest that phosphodiesterases (PDEs), a class of enzymes that selectively hydrolyses cyclic nucleotides, play a key role in determining cAMP signalling specificity to a number of PKA substrates in the heart. Phospholemman (PLM) is a phosphoprotein that is physically and functionally coupled to the cardiac Na/K-ATPase (NKA). The latter provides the primary route of Na extrusion from mammalian cardiac myocytes, thereby contributing to the maintenance of transmembrane ion gradients and the indirect control of myocardial contractility. The work described in this thesis investigated whether individual cardiac PDE subtype(s) are responsible for modulating cAMP signalling to PLM and the NKA in adult rat ventricular myocytes (ARVMs). The use of selective inhibitors for PDE subtypes 2-4 in biochemical assays identified PDE4 as the predominant PDE involved in modulating PLM phosphorylation at Ser68, the consensus PKA site, in the presence of the β-AR agonist isoprenaline hydrochloride (ISO). The augmentation of PLM-Ser68 phosphorylation was accompanied by a corresponding increase in intracellular cAMP accumulation, indicating that PDE4 selectively modulates downstream PKA activity via cAMP hydrolysis. In contrast, ISO-mediated PDE2 inhibition was found to induce the greatest increase in NKA activity, suggesting that upon neurohormonal stimulation, PDE2 may modulate NKA function via a mechanism independent of PKA phosphorylation of PLM. As PDE2 activity comprises a relatively low proportion of total cardiac PDE activity in the rat heart, our findings suggest that the actions of PDE2 may be compartmentalised. A recombinant adenovirus encoding a novel fluorescence resonance energy transfer (FRET)-based cAMP sensor, PLM-Epac1-camps, was generated to enable direct monitoring of cAMP levels in the vicinity of PLM/NKA in ARVMs. Using fluorescence confocal microscopy, PLM-Epac1- camps was observed to be predominantly targeted to the perinuclear regions of the myocytes. However, preliminary ratiometric FRET measurements using a dual-photometer setup demonstrated a robust response to intracellular cAMP elevation with forskolin. Collectively, our findings suggest that further characterisation of this methodology could provide insights into whether PDE2 activity locally regulates cAMP signalling to the NKA in a compartmentalised manner during β-AR stimulation.
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