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 (cGK1?) 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.
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. If successful in the animal model, this genome editing could be translated as a new therapy for Veterans with hypertension- the number one preventable cause of death in this population.
