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 (cGK1?) activation of MP and vasorelaxation. This vasodilator pathway may also be activated by ROS mediated oxidation of cGK1? 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.

Public Health Relevance

The proposed experiments will use the method of ?precision genome editing? to test a novel strategy for lowering of blood pressure in animal models of hypertension. This editing causes a shift in naturally occurring enzyme isoforms in the blood vessel smooth muscle cells that is predicted to make them more sensitive to the signals that relax the muscle and lower blood pressure. This strategy will be tested in animals exposed to stressors throughout their life course. If successful in these animal models this genome editing could be translated as a new therapy for human hypertension, the number one preventable cause of death in the world.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hypertension and Microcirculation Study Section (HM)
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OH, Youngsuk
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University of Maryland Baltimore
Internal Medicine/Medicine
Schools of Medicine
United States
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