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Acute regulation of the cardiac Na pump by extracellular stimuli is crucial in allowing the heart to match its output to systemic requirements. Phospholemman (PLM) regulates the cardiac Na pump. Unphosphorylated PLM inhibits the pump, and PLM phosphorylation causes pump activation. Phospholemman is also palmitoylated at cysteines 40 and 42, which inhibits the pump, and glutathionylated at cysteine 42, which relieves oxidative inhibition of the pump. Since PLM may be palmitoylated and glutathionylated at the same cysteine, we investigated competition between these post-translational modifications during oxidative and metabolic stress in freshly dispersed adult rat ventricular myocytes (ARVM, isolated from adult male Wistar rats) and FT293 cells stably expressing wild type (WT), C40A and C42A PLM. Palmitoylation was measured by resin-assisted capture of acylated proteins followed by quantitative immunoblotting, and glutathionylation by streptavidin affinity purification after loading cells with biotinylated glutathione ethyl ester. Data are presented as mean±SEM, with differences analysed by t-test. Acute exposure of ARVM and FT293 cells expressing wild type PLM to hydrogen peroxide (1μM-10mM, 30min at 37°C) was without effect on PLM palmitoylation. Metabolic stress was induced in FT293 cells expressing WT PLM by 18 hours culture in media supplemented with glucose (HG, 25mM) and / or palmitic acid (HP, 0.4mM). Although HG or HP alone were without effect on PLM palmitoylation, combined HG/HP caused a substantial reduction in PLM palmitoylation (5.7±2.4 fold reduction compared to untreated cells, n=4, p<0.05). Glutathionylated PLM was undetectable in untreated FT293 cells expressing WT PLM, but readily detectable following 18 hours HG/HP culture. In order to identify which palmitoylation site in PLM is susceptible to metabolic stress, FT293 cells expressing palmitoylation site mutants were cultured in HG or HG/HP conditions. HG/HP culture induced a substantial reduction in palmitoylation of C40A PLM (3.0±0.8 fold reduction compared to untreated cells, n=3, p<0.05), but was without effect on the palmitoylation of C42A PLM. In conclusion, metabolic but not acute oxidative stress promotes glutathionylation of PLM cysteine 42 with a concomitant reduction in palmitoylation at this site. Metabolic stress may therefore influence Na pump activity by promoting glutathionylation over palmitoylation of PLM. The cellular outcome of HG/HP stress is mitochondrial free radical production, yet exogenous hydrogen peroxide is without effect on PLM palmitoylation, so the subcellular location at which oxidising species are generated or the duration of an oxidant stress are important determinants of the balance between PLM palmitoylation and glutathionylation. [ABSTRACT FROM AUTHOR]

Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the sodium pump-related calcium ATPase SERCA, PLM is reported to oligomerise. We investigated sub-populations of PLM in adult rat ventricular myocytes based on phosphorylation status. Calcium-tolerant ventricular myocytes (ARVM) were isolated from adult male Wistar rats (200-250g) by retrograde perfusion of collagenase in the Langendorff mode. Myocytes were left to recover for 2 hours at 35°C before experiments. Coimmunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at serine 63 (S63), and one, associated with the pump, both phosphorylated at serine 68 and unphosphorylated. Phosphorylation of PLM at S63 following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localised to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for S63- phosphorylated PLM, and crosslinking reagents also failed to identify any partner proteins for this pool. The sodium pump α subunit co-immuno-precipitated with PP2Afrom ARVM lysates. Since PLM-S63 is the only phosphorylation site in PLM dephosphorylated by PP2A, this likely explains the lack of S63-phosphorylated PLM associated with the pump. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, S63- phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, therefore confirming the existence of PLM multimers. Hence we report the existence of a subpopulation of PLM that interacts only with other PLM molecules and not with the Na pump, with a unique phosphorylation status driven by differential proximity of protein phosphatases. Like phospholamban regulation of SERCA, PLM exists as a sodium pump inhibiting monomer and an un-associated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases, rather than a direct effect of phosphorylation on PLM association with the pump. [ABSTRACT FROM AUTHOR]

Phospholemman (PLM) regulates the cardiac Na pump. Unphosphorylated PLM inhibits the pump, and PLM phosphorylation at S63 and T69 (by PKC), and S68 (by PKA or PKC) causes pump activation. Abnormal phosphorylation of PLM is well established in cardiac hypertrophy and failure. We investigated whether genetic polymorphisms in PLM underlie left ventricular hypertrophy (LVH) by sequencing PCR-amplified PLM exons from anonymised genomic DNA from the Go-DARTS database. Inclusion criteria were age <55, systolic blood pressure <140mmHg, BMI <30kgm-2, and moderate-severe LVH according to echocardiography. 2 out of 43 individuals possessed the same non-synonymous missense coding variant: a C to T transition in position 1 of the codon encoding arginine 70 in PLM exon 7, resulting in the mutation R70C. The R70C PLM phenotype was investigated by expressing wild type (WT) and R70C PLM in HEK293 cells. Phosphorylation induced by activating PKA (10μM forskolin) and PKC (300nM PMA) was measured by phosphospecific immunoblotting, and expressed relative to WT or R70C-expressing cells treated with vehicle. Phosphorylation of WT and R70C PLM by PKA at PLM-S68 was unchanged (2.0±0.3 fold increase over vehicle-treated forWT, 1.8±0.1 fold increase for R70C, mean±SEM, n=4). PKC-induced phosphorylation of PLM-S63 was also unchanged, but R70C PLM was phosphorylated less than WT at S68 following activation of PKC (1.8±0.2 for WT, 1.0±0.2 for R70C, n=4, p<0.05, t-test), suggesting mutation of the arginine at position +2 to S68 had altered this consensus PKC phosphorylation site. We also measured cell surface localisation (membrane-impermeable biotinylation reagents) and palmitoylation (resin-assisted capture of acylated proteins) of WT and R70C PLM.A greater fraction of R70C PLM than WT was found at the cell surface (R70C 3.8±2 fold more enriched than WT, n=3), suggesting a proposed ER retention motif in PLM was also disrupted. A greater fraction of R70CPLM was palmitoylated than WT (53±10% vs 29±5%, n=4, p<0.05), possibly due to this enhanced traffic to the cell surface. Rs61753924, the single nucleotide polymorphism (SNP) underlying PLM R70C, occurs at 0.1 % frequency in the 1000 Genomes Project. We investigated the frequency of this SNP in LVH case and non-LVH controls: 19 out of 393 LVH cases (4.8%) and 10 out of 266 (3.7%) controls were positive for rs61753924 (odds ratio = 1.14, p>0.05). In conclusion, PLM R70C shows reduced phosphorylation at S68 by PKC. In vivo PLM is significantly phosphorylated at rest by PKC, so the reduced phosphorylation and enhanced cell surface localisation and palmitoylation of R70C PLM may lead to reduced Na efflux via the pump. Preliminary analysis indicates that the presence of SNP rs617539241 is not a risk factor for the development of LVH, but is enriched in the local population. [ABSTRACT FROM AUTHOR]