Precision Editing of Myosin Phosphatase for Vasodilator Sensitization in Hypertension

项目摘要 高血压是一种地方病，尽管血管扩张剂和利尿剂治疗仍占世界范围内的大部分 心血管发病率（心力衰竭、中风）和死亡率。本课题主要研究肌球蛋白磷酸酶（Myosin Phosphatase，MP） 其通过去磷酸化肌球蛋白引起平滑肌松弛。MP是缩肌和扩张肌的靶点 调节血管张力从而控制血压的信号通路。我们提出了一个模型， MP调节亚基Mypt 1外显子24（E24）的选择性剪接调节血管平滑肌敏感性 NO/cGMP介导的血管舒张。包含31 nt E24使阅读框架移位，从而编码C- 缺乏cGMP依赖性激酶（cGK 1 α）激活所需的亮氨酸拉链（LZ）基序的末端序列 MP和血管舒张。这一血管扩张通路也可能被ROS介导的cGK 1 α氧化激活 及其下游靶标，包括MP。高血压的血管阻力增加可能部分是由于 降低NO的生物利用度和增加ROS的产生，减少血管舒张信号传导和增加 血管阻力在这里，我们提出测试Mypt 1 E24的精确编辑将减少 血管对血流的阻力和永久性降低高血压患者的血压（BP）。要求1 使用A）Cre-Lox B）Crispr/Cas9的腺相关病毒递送的补充方法，以获得精确度 编辑E24以测试这种方法是否可以逆转高血压AngII模型中的血管功能障碍。它 比较了高血压病的原始预防和治疗方法。分子测定 将确定NO/cGMP/ROS如何激活MP，测试一种新的2池“刹车和加速器”模型， 在高血压中，在MP控制中扩张器和收缩器信号的整合，从而控制血压。 高血压素质可能在生命早期开始，这一点可以通过终生血压跟踪来证明 当然了终生血压与心血管结局的关系最为密切， 并不能使心血管疾病的发病率和死亡率正常化。这些为研究下列问题提供了令人信服的理由： 高血压的规划及其在生命早期的基本预防。我们已经证明， Mypt 1的E24+（“cGMP抗性”）同种型在青春期作为动脉成熟的一部分发生， 与血管收缩功能和血压升高一致。这一过程被早期的生活压力加速， 是高血压发生的重要危险因素。目标2将测试E24的精确编辑能力 在生命早期，以减轻早期或整个生命过程中压力对动脉功能的有害影响， 编程.它还将测试是否通过在生命早期精确编辑E24来预防血管功能障碍 比高血压病好的时候更有效。我们预计，这种新的战略， 精确编辑Mypt 1选择性外显子24，通过改变MP同工酶池以有利于血管扩张剂 信号传导，将导致血管扩张剂敏化和整个生命过程中的低血压。如果成功， 实验策略具有很高的潜力，作为一种新的治疗人类的翻译。

Project Summary High blood pressure is endemic, and despite vasodilator and diuretic therapy still accounts for much world-wide cardiovascular morbidity (heart failure, stroke) and mortality. Our studies focus on Myosin Phosphatase (MP) which by de-phosphorylating myosin causes smooth muscle relaxation. MP is the target of constrictor and dilator signaling pathways that regulate vascular tone and thereby control BP. We have proposed a model in which alternative splicing of Exon 24 (E24) of the MP regulatory subunit Mypt1 tunes vascular smooth muscle sensitivity to NO/cGMP-mediated vasorelaxation. Inclusion of the 31 nt E24 shifts the reading frame, thus coding for a C- terminal sequence lacking the leucine zipper (LZ) motif required for cGMP-dependent kinase (cGK1α) activation of MP and vasorelaxation. This vasodilator pathway may also be activated by ROS mediated oxidation of cGK1α and its downstream targets including MP. The increased vascular resistance of hypertension may in part be due to reduced bio-availability of NO and increased ROS generation reducing vasodilator signaling and increasing vascular resistance. Here we propose to test the hypothesis that precision editing of Mypt1 E24 will reduce vascular resistance to blood flow and permanently lower blood pressure (BP) in hypertension. Aim 1 uses complementary approaches of A) Cre-Lox B) Adeno-Associated Viral delivery of Crispr/Cas9, for precision editing of E24 to test if this approach can reverse vasodysfunction in the AngII model of hypertension. It compares approaches of primordial prevention vs treatment after hypertension is established. Molecular assays will determine how NO/cGMP/ROS activate MP, testing a novel 2-pool “brake and accelerator” model for the integration of dilator and constrictor signals in the control of MP, and thus blood pressure, in hypertension. The hypertensive diathesis may initiate early in life as evidenced by tracking of BP throughout the life course. Lifetime BP is most strongly related to cardiovascular outcomes, and effective lowering of BP in maturity does not normalize cardiovascular morbidity and mortality. These provide compelling rationale for the study of programming of hypertension and its primordial prevention early in life. We have shown that the switch to the E24+ (“cGMP resistant”) isoform of Mypt1 occurs during adolescence as part of arterial maturation and concordant with increasing vasoconstrictor function and BP. This process is accelerated by early life stress, an important risk factor in the development of hypertension. Aim 2 will test the ability of precision editing of E24 early in life to mitigate the deleterious effect of stress early, or throughout, the life course, on arterial function and programming. It will also test if primordial prevention of vasodysfunction via precision editing of E24 in early life is more effective as compared to after hypertension is well established. We expect that this novel strategy of precision editing of the Mypt1 alternative exon 24, by shifting the MP isozyme pools to favor vasodilator signaling, will cause vasodilator sensitization and lower BP throughout the life course. If successful this experimental strategy has high potential for translation as a new therapeutic in humans.