Precision Editing of Myosin Phosphatase for Vasodilator Sensitization in Hypertension

项目摘要 高血压是地方性疾病，尽管血管扩张剂和利尿剂治疗在世界范围内仍占很大比例。 心血管发病率(心力衰竭、中风)和死亡率。我们的研究重点是肌球蛋白磷酸酶(MP) 它通过去磷酸化肌球蛋白导致平滑肌松弛。MP是缩窄器和扩张器的目标 调节血管张力从而控制血压的信号通路。我们已经提出了一个模型，在这个模型中 MP调节亚单位Mypt1外显子24(E24)的选择性剪接调节血管平滑肌敏感性 NO/cGMP介导的血管松弛作用。包括31个核苷酸的E24转移了阅读框，从而编码了C- 末端序列缺乏cGMP依赖的激酶(cGK1α)激活所需的亮氨酸拉链(LZ)基序 MP和血管松弛的作用。这种血管扩张途径也可能被ROS介导的cGK1α氧化所激活 其下游目标包括甲氧西林。高血压患者血管阻力增加的部分原因可能是 减少NO的生物利用度，增加ROS的产生，减少血管扩张信号转导和增加 血管阻力。在这里，我们建议测试Mypt1 E24的精确度编辑将降低的假设 高血压患者的血管血流阻力和永久性的血压降低。目标1 使用A)Cre-Lox B)腺相关病毒传递Crispr/Cas9的互补方法，以提高精确度 编辑E24以测试这种方法是否可以逆转Angii高血压模型中的血管功能障碍。它 比较高血压建立后的原始预防和治疗方法。分子分析 将确定NO/cGMP/ROS如何激活MP，测试用于 扩张器和收缩信号在MP控制中的整合，从而在高血压中控制血压。 高血压素质可能在生命早期就开始了，这一点可以通过一生中对血压的跟踪来证明 当然了。终生血压与心血管结果密切相关，成熟期有效降低血压 不会使心血管发病率和死亡率正常化。这些都为研究提供了令人信服的理由 生命早期高血压的规划及其原始预防。我们已经证明，切换到 Mypt1的E24+(“cGMP抵抗”)亚型在青春期出现，是动脉成熟和 与血管收缩功能和血压升高相一致。这一过程因早期的生活压力而加速， 高血压发病的重要危险因素。目标2将测试E24的精确编辑能力 在生命早期或在整个生命过程中减轻压力对动脉功能的有害影响 编程。它还将测试早期是否通过精确编辑E24来预防血管功能障碍 与高血压得到证实后相比，更有效。我们预计，这一新颖的战略 通过改变MP同工酶库以支持血管扩张剂，精确编辑Mypt1替代外显子24 信号传递，会导致血管扩张剂敏化，并在整个生命过程中降低血压。如果成功，请执行此操作 实验策略具有很高的潜力转化为人类的一种新的治疗方法。

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.