Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans

Background: While it has long been recognized that smooth muscle Na/K ATPase (NKA) modulates vascular tone and blood pressure (BP), the role of its accessory protein phopholemman (PLM) has not been characterized. The aim of this study was to test the hypothesis that PLM phosphorylation regulates vascular tone in vitro and this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in man. Methods: Mouse studies: PLM knock-in mice (PLM3SA), in which PLM is rendered unphosphorylatable, were used to assess the role of PLM phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type (WT) and transgenic mice. Human studies: We searched human genomic databases for mutations in PLM in the region of the phosphorylation sites and performed analyses within two human data cohorts (UK Biobank and GoDARTS) to assess the impact of an identified SNP on BP. This SNP was expressed in HEK cells and its effect on PLM phosphorylation determined using Western Blotting. Results: PLM phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of PLM phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to PE both in vitro and in vivo. In ageing WT mice PLM was hypophosphorylated and this correlated with the development of ageing-induced essential hypertension. In man we identified a non-synonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in PLM. In HEK cells the R70C mutation prevented PLM phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. Conclusions: These studies demonstrate the importance of PLM phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for ageing-induced essential hypertension in man