From the University Paris-Sud, Faculté de Médecine, Université Paris-Saclay, Le Kremlin Bicêtre, France (M.L., V.C., A.B., M.K.N., A.H., C.R.-M., O.M., B.G., D.M., F.P., M. H., F.A.).
Assistance Publique Hôpitaux de Paris, Service de Pneumologie, Centre de Référence de l'Hypertension Pulmonaire, Hôpital Bicêtre, Le Kremlin Bicêtre, France (M.L., V.C., A.B., M.K.N., A.H., C.R.-M., O.M., B.G., D.M., F.P., M.H., F.A.).
Circ Res. 2019 Sep 13;125(7):678-695. doi: 10.1161/CIRCRESAHA.119.314793. Epub 2019 Jul 26.
Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K channel, have been identified in pulmonary arterial hypertension patients.
We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature.
Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from -mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. -mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. -mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of -mutated rats were characterized by overactivation of ERK1/2 (extracellular signal-regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, -mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, -mutated rats developed age-dependent PH associated with low serum-albumin concentration.
We established the first -mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.
肺动脉高压是一种严重的致命心肺疾病。已经在肺动脉高压患者中鉴定出编码外向整流钾通道的（钾通道亚家族 K 成员 3）基因的功能丧失突变。
我们已经证明，KCNK3 功能障碍与遗传性和非遗传性肺动脉高压以及实验性肺动脉高压（PH）都有关。最后，KCNK3 在小鼠肺血管中没有功能。
使用 CRISPR/Cas9 技术，我们在 基因的外显子 1 中产生了 94 个碱基对的移框缺失，并在电生理学、超声心动图、血液动力学、形态学、细胞和分子水平上对这些大鼠进行了特征描述，以破译与 KCNK3 缺失相关的细胞机制。使用膜片钳技术，我们通过证明从 -突变大鼠新鲜分离的肺动脉平滑肌细胞中不存在 KCNK3 电流来验证我们的转基因策略。在 4 个月大时，超声心动图参数显示肺动脉加速时间缩短，同时右心室收缩压升高。与野生型大鼠相比，暴露于单克隆毒素或慢性低氧后，-突变大鼠更易发生 PH。-突变诱导肺远端新生肌化和血管周围细胞外基质激活。-突变大鼠的特征是 ERK1/2（细胞外信号调节激酶 1/2）、AKT（蛋白激酶 B）、SRC 的过度激活以及 HIF1-α（缺氧诱导因子-1α）、survivin 和 VWF（血管性血友病因子）的过度表达。与质膜去极化相关，内皮型一氧化氮合酶表达减少和内皮衍生超极化因子脱敏，-突变大鼠表现出肺血管收缩的倾向和严重丧失西地那非诱导的肺血管松弛。此外，我们还显示右心室心肌细胞兴奋性发生强烈改变。最后，-突变大鼠出现与血清白蛋白浓度低相关的年龄依赖性 PH。
我们建立了首个 -突变大鼠 PH 模型。我们的结果证实，KCNK3 功能丧失是肺动脉高压发病机制中的关键事件。该模型为理解 PH 的起始机制以及在 PH 背景下测试具有生物学相关性的治疗分子提供了新的机会。
