The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice
The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice
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DOI：
10.1096/fj.201700794r
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发表时间：
2018-04-01
期刊：
影响因子：
4.8
通讯作者：
Hori, Masatoshi
中科院分区：
文献类型：
--
作者：
Yang, Qunhui;Fujii, Wataru;Hori, Masatoshi
蛋白激酶C增强的磷酸化依赖的蛋白磷酸酶抑制蛋白1(CPI-17)是一种内源性肌球蛋白磷酸酶抑制蛋白，被认为是钙增敏收缩装置的关键分子。在这里，我们使用聚集的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9来产生CPI-17缺陷[基因敲除(KO)]和苏氨酸38(T38)磷酸抗性小鼠[苏氨酸突变为丙氨酸(TA)]，然后研究CPI-17对体外血管收缩和体内平均血压(MBP)的影响。在分离的胸主动脉上，佛波醇12，13-二丁酸酯可引起野生型(WT)小鼠持续收缩，而TA或KO小鼠则无收缩。在转基因小鼠和WT小鼠中，高浓度KCl溶液引起的收缩没有区别。相反，在两个突变品系中，苯肾上腺素(PE)诱导的收缩明显小于WT小鼠，并伴随着低水平的肌球蛋白磷酸化，这表明至少部分PE诱导的收缩是由T38处CPI-17的磷酸化调节的。最后，利用无线电遥测技术研究了CPI-17在血压调节中的生理作用。在两只转基因小鼠中，MBP显著降低，即使心率代偿性增加。总之，我们首次建立了KO和对CPI-17具有结构性磷酸盐抗性的小鼠模型。位于T38的P-CPI-17可能由PKC调控，对维持体内血管的收缩能力和血压可能很重要。
PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [threonine mutant into alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12, 13-dibutyrate induced a sustained contraction of wild-type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution-induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)-induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE-induced contraction is regulated by phosphorylation of CPI-17 at T38. Finally, the physiologic role of CPI-17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho-resistant mouse models of CPI-17 for the first time. p-CPI-17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo.