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) 是一种地方病，尽管采用血管扩张剂和利尿剂治疗，但仍占很大比例。 退伍军人群体的心血管发病率（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。议员是 大多数收缩肌和扩张肌信号通路的靶标，可调节血管张力，从而控制血压。超过 经过多年的研究，我们提出了一个模型，其中 MP 监管的外显子 24 (E24) 的选择性剪接 Mypt1 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 会改变阅读框，从而编码缺少 C- 的 Mypt1 同工型 MP 和 cGMP 依赖性激酶 (cGK1α) 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 的抑制亚基 CPI17 是抑制信号的关键目标 MP 从而引起血管收缩。高血压导致血管阻力增加的部分原因是 NO 生物利用度降低，血管舒张信号传导减少，神经体液激活增加 血管收缩信号传导。 在这里，我们建议在高血压动物模型中测试涉及精确编辑的新策略 MP 调节和抑制亚基旨在改变收缩肌和扩张肌信号的平衡 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的敏感性 MP 调节亚基 Mypt1 外显子 24 (E24)。目标 3 将通过精确度测试血管收缩剂脱敏作用 MP 抑制亚基 CPI17 的编辑。 目标 1 将开发 E24 Crispr/Cas9 编辑（删除）的腺相关病毒传递，以测试这是否有效 该方法可以逆转 AngII 小鼠高血压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，该模型可实现对 相同的目标，并比较高血压发病后的原始预防和治疗方法。 Aim 3 将开发 CPI17 的 Crispr/Cas9 编辑的腺相关病毒传递，以测试这种方法是否可以 抑制 AngII 小鼠高血压模型中血管收缩信号的增加。 分子分析将确定扩张器和收缩器信号整合的机制 MP 在正常和高血压动物中控制血压，以及如何通过精确编辑改变这种平衡 MP 亚基。这些实验具有向退伍军人转化的巨大潜力，将测试一部小说 用于永久降低血压的血管扩张剂增敏/血管收缩剂脱敏策略，以及 将定义信号汇聚到 MP 上以引起模型中血管功能障碍的机制 高血压。