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，尽管血管舒张剂，dis缩剂和利尿治疗，但仍在说明很多 在退伍军人人群中，心血管疾病（心力衰竭，中风）和死亡率。 肌球蛋白磷酸酶（MP），通过去磷酸化肌球蛋白会导致平滑肌松弛。 大多数收缩和扩张器信号通路，这些途径会逐渐使用。 多年研究我们已经支撑了一个模型，其中MP调节的外显子24（E24）的替代剪接 亚基MYPT1调节血管平滑肌敏感对一氧化氮（NO）/ CGMP介导的血管汇总。 包括31 NT E24的包含移动框架，从而编码缺少C-的MyPT1的同工型 MP和 其他其他人表明，MP的不便亚基CPI17是监视器的关键目标 MP和THEBY引起的血管收缩。 无减少血管扩张器信号传导的生物可用性降低，而神经肿瘤激活增加 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新型策略的动物模型。 MP调节器和抑制亚基旨在转移收缩和扩张器信号的平衡 为了降低血管电阻和BP。 MP监管亚基MYPT1外显子24（E24）。 MP抑制亚基CPI17的编辑。 AIM 1将开发与E24的CRISPR/CAS9编辑（删除）的腺相关病毒delially，以测试是否这样 方法可以在高血压的Angii小鼠模型中逆转血管扩展。 AIM 2将使用我们最近开发和验证的CRE-LOX模型，该模型可实现强大的编辑。 相同的目标，并比较建立高血压后原始预防与治疗方法的方法。 AIM 3将开发CRISPR/CAS9的CPI17的腺相关病毒传递，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。 分子测定Willmine机制，通过该机制通过将扩张器和收缩器信号整合到 在正常和高血压动物中控制BP的MP，以及通过呈现编辑如何改变这种弹力 这些实验的MP亚基，具有很高的翻译为退伍军人 血管舒张敏化的策略/血管收缩剂量的每个名称 将定义机制，通过该机制，信号在MP上汇合以引起血管函数 高血压。