British Heart Foundation Centre of Research Excellence, King's College London, United Kingdom (A.B., S.T., O.P., O.R., D.S.-T., M.J.S.). Clinical Pharmacology, The William Harvey Research Institute (O.P., H.R.W., M.J.C.), National Institute for Health Research, Biomedical Research Centre (H.R.W., M.J.C.), Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom. Department of Pharmacology, University of Oxford, United Kingdom (H.A.L.L., K.A.D., C.J.G.). Medicines Monitoring Unit, School of Medicine (A.D.), Division of Cardiovascular and Diabetes Medicine (C.N.A.), University of Dundee, United Kingdom. Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (J.V.W., J.H., W.F.).
Circulation. 2021 Mar 16;143(11):1123-1138. doi: 10.1161/CIRCULATIONAHA.119.040557. Epub 2020 Dec 18.
Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans.
In mouse studies, phospholemman knock-in mice (PLM; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman 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 and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting.
Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men.
These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.
尽管平滑肌 Na/K ATPase 调节血管张力和血压（BP）早已得到认可，但它的辅助蛋白磷酸烯醇式丙酮酸羧激酶同工酶（phospholemman）的作用尚未得到描述。本研究旨在检验以下假说，即磷酸烯醇式丙酮酸羧激酶同工酶磷酸化调节体外血管张力，并且该机制在体内实验模型和人类中调节血管功能和 BP 方面发挥重要作用。
在小鼠研究中，使用磷酸烯醇式丙酮酸羧激酶同工酶敲入小鼠（PLM；磷酸烯醇式丙酮酸羧激酶同工酶 [FXYD1] 中丝氨酸 63、68 和 69 上的 3 个磷酸化位点突变为丙氨酸，从而使磷酸烯醇式丙酮酸羧激酶同工酶不可磷酸化）来评估体外主动脉和肠系膜血管中磷酸烯醇式丙酮酸羧激酶同工酶磷酸化在 wire myography 和膜电位测量中的作用。使用多普勒流量和遥测术在年轻（14-16 周）和年老（57-60 周）野生型和转基因小鼠中评估体内 BP 和局部血流。在人类研究中，我们在磷酸烯醇式丙酮酸羧激酶同工酶的磷酸化位点区域的人类基因组数据库中搜索突变，并在 2 个人类队列（英国生物银行和 GoDARTS[泰赛德遗传学糖尿病审计和研究]）中进行分析，以评估鉴定的单核苷酸多态性对 BP 的影响。该单核苷酸多态性在人胚肾细胞中表达，并使用 Western blot 确定其对磷酸烯醇式丙酮酸羧激酶同工酶磷酸化的影响。
磷酸烯醇式丙酮酸羧激酶同工酶丝氨酸 63 和丝氨酸 68 的磷酸化限制了对苯肾上腺素的血管收缩反应。该作用被哇巴因阻断。PLM 小鼠中磷酸烯醇式丙酮酸羧激酶同工酶磷酸化的预防极大地增强了体外和体内对苯肾上腺素的血管反应。在衰老的野生型小鼠中，磷酸烯醇式丙酮酸羧激酶同工酶的磷酸化程度降低，这与衰老引起的原发性高血压的发生有关。在人类中，我们鉴定出一个非同义编码变异，单核苷酸多态性 rs61753924，导致磷酸烯醇式丙酮酸羧激酶同工酶中的 R70C 取代。在人胚肾细胞中，R70C 突变阻止了磷酸烯醇式丙酮酸羧激酶同工酶丝氨酸 68 的磷酸化。该变体的罕见等位基因与中年男性的 BP 升高显著相关。
这些研究表明磷酸烯醇式丙酮酸羧激酶同工酶磷酸化在调节血管张力和 BP 中的重要性，并提示了一种新的机制和治疗靶点，用于治疗人类衰老引起的原发性高血压。
