Phenotypic characterisation of the PLM3SA mouse expressing unphosphorylatable phospholemman

Cardiac Na+/K+ ATPase plays a pivotal role in maintaining the Na+ transmembrane gradient which is essential for normal cardiac function. Elevation of intracellular Na+ ([Na+]i) is a key contributor to contractile and electrical dysfunction in a variety of pathologies. Phospholemman (PLM), is the cardiac specific member of the FXYD family of small membrane spanning proteins, and forms a complex with Na+/K+ ATPase pump. PLM regulates the pump by exerting a tonic inhibition which is relieved by PKA or PKC phosphorylation at 3 serine (Ser)/threonine (Thr) residues in its cytoplasmic tail (Ser63, Ser68, Thr/Ser69). Phosphorylation at any of these sites results in disinhibition of the pump and even active stimulation. The work described in this thesis investigates the role of PLM in regulating the Na+/K+ ATPase and the subsequent effect on [Na+]i. A new mouse model, PLM3SA, has been created by mutating all 3 Ser residues to alanine (Ala) rendering the protein unphosphorylatable. This inability to phosphorylate PLM may cause [Na+]i overload, contractile dysfunction and cardiac arrhythmias. The aim of this thesis was to investigate the effects of unphosphorylatable PLM on Na+/K+ ATPase regulation and [Na+]i by characterising the PLM3SA mouse phenotype using biochemical and Langendorff methodology under basal conditions and during β-receptor stimulation. The measurement of [Na+]i using flame photometry and 23Na NMR completed the work. Biochemical studies confirmed the successful mutation of the PLM protein and absence of Ser63, Ser68 and Ser69 phosphorylation sites. Mutated PLM was found to remain co-localised with the Na+/K+ ATPase α1 subunit in the membrane. Expression of total PLM was decreased in PLM3SA hearts, however all Na+/K+ ATPase subunit expression remained unchanged. Comparison of paced basal contractility in the PLM-WT and PLM3SA hearts revealed that PLM3SA hearts have significantly greater contractility, consistent with an inhibited pump and increased [Na+]i. Arrhythmia quantification revealed no differences in scores between genotypes suggesting [Na+]i was not detrimental under these conditions. Force-frequency relationship (FFR) studies were consistent with a high [Na+]i in both genotypes. However, the lack of difference between WT and PLM3SA hearts suggests that changes in [Na+]i in response to pacing do not substantially differ between genotypes. β-receptor stimulation by isoprenaline (ISO) perfusion was investigated to highlight potential changes in cardiac function between unphosphorylatable PLM in PLM3SA hearts compared with Abstract iii WT. Contractile data however revealed no differences suggesting that PKA activation of other key excitation-contraction coupling proteins dominate the contractile response to ISO. Direct measurement of [Na+]i using flame photometry and 23Na NMR showed no differences in intracellular Na+ concentration in PLM3SA hearts compared with WT under basal conditions. Quantification of [Na+]i by 23Na NMR using the shift reagent, Tm(DOTP)5-, gave values for PLM3SA hearts of 12.7±2.5mM, and 14.49±3.1mM for PLM-WT hearts. β-receptor stimulation, pacing and ouabain effects on [Na+]i were also investigated and ouabain significantly increased [Na+]i compared to baseline in both PLM3SA and WT hearts to a similar extent. The work described in this thesis investigates the role of unphosphorylatable PLM on [Na+]i in PLM3SA hearts revealing findings that show:  No effect on [Na+]i under basal conditions  No evidence for differences in basal Na+/K+ ATPase activity - based on rubidium (Rb+) uptake flame photometry studies  No evidence for a role of PLM phosphorylation in control of Na+ at high pacing rates or in the presence of ISO. However, these conclusions may reflect technical limitations of the experimental approaches used.