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

肌球蛋白磷酸酶的精确编辑作为高血压患者血管扩张剂增敏和降血压的新方法

• 批准号: 10265343
• 负责人: Steven A. Fisher
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10265343
• 关键词: Aftercare; Alternative Splicing; Angiotensin II; Animal Model; Animals; Biological Assay; Biological Availability; Blood Pressure; Blood Vessels; Blood flow; C-terminal; CRISPR/Cas technology; Cardiovascular Physiology; Cardiovascular system; Cause of Death; Code; Cre-LoxP; Cyclic GMP; Dilator; Disease; Diuretics; Enzymes; Equilibrium; Exons; Genes; Health; Health system; Heart failure; Hypertension; Knowledge; Leucine Zippers; Longevity; Mediating; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle; Muscle relaxation phase; Myosin ATPase; Myosin S-1; Nitric Oxide; Peripheral Resistance; Phosphotransferases; Population; Prevention; Protein Isoforms; Reading Frames; Signal Pathway; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Stroke; Tamoxifen; Testing; Therapeutic; Translating; Translations; United States; Vascular Smooth Muscle; Vascular resistance; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Veterans; Viral; blood pressure reduction; blood pressure regulation; desensitization; experimental study; genome editing; hypertension treatment; improved; in vivo; military veteran; mortality; mouse model; myosin phosphatase; novel; novel strategies; novel therapeutics; personalized approach; response; success; vasoconstriction

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.

高血压（BP）是地方性的，尽管有血管扩张剂和利尿剂治疗， 退伍军人人群的心血管发病率（心力衰竭、中风）和死亡率。我们的研究集中在 肌球蛋白磷酸酶（MP），通过去磷酸化肌球蛋白引起平滑肌松弛。MP是 调节血管张力从而控制血压的大多数收缩和扩张信号通路的靶点。超过 经过多年的研究，我们提出了一个模型，其中MP的外显子24（E24）的选择性剪接调节 Mypt 1亚基调节血管平滑肌对一氧化氮（NO）/cGMP介导的血管舒张的敏感性。 包含31 nt E24使阅读框移位，从而编码缺少C-末端的Mypt 1同种型。 MP的cGMP依赖性激酶（cGK 1 α）激活所需的末端亮氨酸拉链（LZ）基序， 血管舒张其他人已经表明MP的抑制性亚基CPI 17是抑制信号的关键靶点， MP，从而引起血管收缩。高血压的血管阻力增加部分是由于 NO的生物利用度降低，血管舒张信号减少，神经体液激活增加 血管收缩信号 在这里，我们提出在高血压动物模型中测试涉及精确编辑的新策略。 MP调节和抑制亚单位旨在改变收缩和扩张信号的平衡， 以降低血管阻力和血压。目标1和2将通过精确编辑测试血管扩张剂致敏性 MP调节亚基Mypt 1外显子24（E24）的表达。Aim 3将通过精密度测试血管收缩剂脱敏 MP抑制亚基CPI 17的编辑。 目标1将开发E24的Crispr/Cas9编辑（缺失）的腺相关病毒递送，以测试这是否 方法可以逆转高血压AngII小鼠模型中的血管功能障碍。 Aim 2将使用我们最近开发和验证的Cre-Lox小鼠模型，该模型实现了对 相同的目标，并比较了高血压病确立后的原始预防与治疗方法。 Aim 3将开发CPI 17的Crispr/Cas9编辑的腺相关病毒递送，以测试这种方法是否可以 抑制高血压AngII小鼠模型中增加的血管收缩信号传导。 分子分析将确定扩张器和收缩器信号通过以下方式整合的机制： MP在正常和高血压动物中控制BP，以及这种平衡如何通过精确编辑改变 MP亚单位的。这些实验，具有很高的潜力翻译为退伍军人，将测试一部小说 血管扩张剂增敏/血管收缩剂脱敏策略，永久性降低血压，以及 将定义信号会聚在MP上导致血管功能障碍的机制， 高血压