Precision Editing of Myosin Phosphatase as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure in Hypertension

高血压(BP)是地方性疾病，尽管有血管扩张剂和利尿剂治疗，但仍占很大比例。 退伍军人的心血管发病率(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)，通过使肌球蛋白去磷酸化而导致平滑肌松弛。国会议员是 大多数收缩和扩张信号通路的靶标，调节血管张力，从而控制血压。完毕 经过多年的研究，我们提出了一种MP调控基因外显子24(E24)的选择性剪接模型 Mypt1亚基调节血管平滑肌对一氧化氮(NO)/cGMP介导的血管松弛的敏感性。 包含31个核苷酸的E24改变了阅读框架，从而编码缺乏C-1的Mypt1的异构体。 端亮氨酸拉链(LZ)基序是激活cGMP依赖的蛋白激酶(cGK1α)所必需的 血管松弛。其他研究表明，MP的抑制亚基CPI17是抑制信号的关键靶点 从而引起血管收缩。高血压患者血管阻力增加的部分原因是 NO生物利用度降低降低血管扩张剂信号转导，增加神经体液激活 血管收缩信号。 在这里，我们建议在高血压的动物模型中测试新的策略，包括精确编辑 MP调节和抑制亚基旨在改变收缩和扩张信号的平衡 以降低血管阻力和血压。AIMS 1和2将通过精确编辑测试血管扩张剂敏化 MP调节亚基Mypt1外显子24(E24)。AIM 3将通过Precision测试血管收缩药脱敏 MP抑制亚基CPI17的编辑。 AIM 1将开发腺相关病毒递送E24的Crispr/Cas9编辑(缺失)以测试是否 可逆转Angii高血压小鼠模型的血管功能障碍。 Aim 2将使用我们最近开发和验证的Cre-Lox鼠标模型，该模型实现了对 相同的目标，并比较高血压建立后的原始预防和治疗方法。 AIM 3将开发Crispr/Cas9编辑CPI17的腺相关病毒递送，以测试这种方法是否可以 抑制血管收缩信号在Angii小鼠高血压模型中的增加。 分子分析将确定扩张器和收缩信号整合的机制 MP在正常和高血压动物的血压控制中的作用，以及这种平衡是如何通过精确编辑改变的 MP亚基的比例。这些实验，很有可能翻译成退伍军人，将测试一部小说 血管扩张剂敏化/血管收缩药脱敏用于永久性降压的策略，以及 将确定信号汇聚在MP上以导致血管功能障碍的机制 高血压。

High blood pressure (BP) is endemic, and despite vasodilator and diuretic therapy, still accounts for much cardiovascular morbidity (heart failure, stroke) and mortality in the veteran population. Our studies focus on Myosin Phosphatase (MP), which by de-phosphorylating myosin causes smooth muscle relaxation. MP is the target of most constrictor and dilator signaling pathways that regulate vascular tone and thereby control BP. Over years of study 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 nitric oxide (NO)/ cGMP-mediated vasorelaxation. Inclusion of the 31 nt E24 shifts the reading frame, thereby coding for an isoform of Mypt1 lacking the C- terminal leucine zipper (LZ) motif required for cGMP-dependent kinase (cGK1α) activation of MP and vasorelaxation. Others have shown that the inhibitory subunit of MP, CPI17, is a key target of signals that inhibit MP and thereby cause vasoconstriction. The increased vascular resistance of hypertension is in part due to reduced bio-availability of NO reducing vasodilator signaling, and neurohumoral activation increasing vasoconstrictor signaling. Here we propose to test in an animal model of hypertension novel strategies involving precision editing of the MP regulatory and inhibitory subunits aimed at shifting the balance of constrictor and dilator signaling in order to lower vascular resistance and BP. Aims 1 and 2 will test vasodilator sensitization via precision editing of MP regulatory subunit Mypt1 Exon 24 (E24). Aim 3 will test vasoconstrictor de-sensitization via precision editing of the MP inhibitory subunit CPI17. Aim 1 will develop Adeno-Associated Viral delivery of Crispr/Cas9 editing (deletion) of E24 to test if this approach can reverse vasodysfunction in the AngII mouse model of hypertension. Aim 2 will use our recently developed and validated Cre-Lox mouse model that achieves robust editing of the same target, and compares approaches of primordial prevention vs treatment after hypertension is established. Aim 3 will develop Adeno-Associated Viral delivery of Crispr/Cas9 editing of CPI17 to test if this approach can suppress the increased vasoconstrictor signaling in the AngII mouse model of hypertension. Molecular assays will determine mechanisms by which dilator and constrictor signals are integrated by MP in the control of BP in normal and hypertensive animals, and how this balance is altered by precision editing of the MP subunits. These experiments, with high potential for translation to Veterans, will test a novel strategy of vasodilator sensitization/vasoconstrictor desensitization for permanent lowering of BP, and will define mechanisms by which signals converge on MP to cause vasodysfunction in models of hypertension.