Precision Editing of Myosin Phosphatase for Vasodilator Sensitization in Hypertension

肌球蛋白磷酸酶的精确编辑对高血压血管舒张剂的敏感性

• 批准号: 9894837
• 负责人: Steven A. Fisher
• 金额: $ 34.76万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-20 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894837
• 关键词: Address; Adolescence; Adolescent; Aftercare; Alternative Splicing; Angiotensin II; Animal Model; Animals; Arteries; Biological Assay; Biological Availability; Birth; Blood Pressure; Blood Vessels; Blood flow; C-terminal; CRISPR/Cas technology; Cardiovascular system; Cause of Death; Code; Cre-LoxP; Cyclic GMP; Development; Dilator; Disease susceptibility; Diuretics; Enzymes; Essential Hypertension; Exons; Exposure to; Generations; Genes; Guide RNA; Heart failure; Human; Hypertension; Isoenzymes; Leucine Zippers; Life; Life Cycle Stages; Mammals; Measures; Mediating; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle; Muscle relaxation phase; Myosin ATPase; Outcome; Pathogenesis; Pathway interactions; Peripheral Resistance; Phosphotransferases; Prevention; Process; Protein Isoforms; RNA Editing; Reactive Oxygen Species; Reading Frames; Resistance; Risk Factors; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Somatic Cell; Stress; Stress Tests; Stroke; Testing; Therapeutic; Time; Translating; Translations; Vascular Smooth Muscle; Vascular resistance; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Viral; Weaning; blood pressure reduction; blood pressure regulation; early life stress; experimental study; genome editing; hypertension prevention; in vivo; innovation; insight; lifetime risk; maternal separation; mortality; myosin phosphatase; novel; novel strategies; novel therapeutics; oxidation; stressor; success

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.

项目摘要 高血压是内在的，dospite血管扩张剂和利尿治疗仍然占全球范围内的 心血管发病率（心力衰竭，中风）和死亡率。我们的研究集中于肌球蛋白磷酸酶（MP） 通过去磷酸化，肌球蛋白会导致平滑肌松弛。 MP是收缩和扩张器的目标 调节血管张力并从而控制BP的信号通路。我们提出了一个模型 MP调节亚基MYPT1调节的外显子24（E24）的替代剪接均具有血管平滑肌灵敏度 无/CGMP介导的血管汇总。包含31 NT E24会改变阅读框，从而编码C- 缺少CGMP依赖性激酶（CGK1α）激活所需的亮氨酸拉链（LZ）基序的末端序列 MP和血管汇总。该血管扩张途径也可以通过ROS介导的CGK1α氧化激活 以及包括MP在内的下游目标。高血压的血管抵抗增加可能部分应 降低了NO的生物可用性和增加ROS产生的增加，减少了血管扩张器信号并增加 血管抗性。在这里，我们建议测试MyPT1 E24的精确编辑的假设 高血压中对血液流量的血管抗性和永久性降低血压（BP）。目标1 使用a）Cre-lox b）CRISPR/CAS9的腺相关病毒传递的完整方法 E24的编辑以测试这种方法是否可以在高血压的Angii模型中逆转血管函数。它 比较建立高血压后原始预防与治疗方法的方法。分子测定 将确定没有/cGMP/ROS如何激活MP，测试了新型的2-pool“制动和加速器”模型 在高血压中，在MP控制中的扩张器和收缩器信号的整合。 高血压素质可能在生命的早期开始 课程。终身BP与心血管结局最密切相关，并且有效地降低了BP的成熟度 不会标准化心血管发病率和死亡率。这些为研究提供了令人信服的理由 高血压及其生命早期的原始预防编程。我们已经证明了转换到 MyPT1的E24+（“ CGMP抗药性”）在青少年期间发生，作为动脉成熟的一部分和 与增加血管收缩功能和BP一致。早期生活压力加速了这个过程 高血压发展的重要危险因素。 AIM 2将测试E24精确编辑的能力 在生命的早期，以减轻压力的有害影响早期或整个生命过程，对动脉功能和 编程。它还将测试通过早期E24的精确编辑来预防血管函数 与高血压之后相比，更有效。我们期望这种新颖的策略 通过移动MP同工酶池以偏爱血管扩张剂，对MYPT1替代外显子24的精确编辑 信号传导将在整个生命过程中引起血管扩张剂的灵敏度和降低的BP。如果成功 实验策略具有很高的转化潜力，可以作为人类的新疗法。