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: 10338049
• 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 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10338049
• 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 control; blood pressure reduction; early life stress; experimental study; genome editing; hypertension prevention; hypertensive; 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.

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