Smooth Muscle‐Specific Deletion of Cullin‐3 Causes Severe Early Onset Hypertension
Authors: Agbor Larry N , Nair Anand R , Wu Jing , Davis Deborah R , Keen Henry L , Quelle Frederick W , McCormick James A , Singer Jeffrey D , Sigmund Curt D 
Published: November 2023

Dominant de novo Cullin‐3 mutations resulting in the skipping of exon 9 (CUL3Δ9) cause severe early onset human hypertension in patients with Pseudohypoaldosteronism type II (PHAII). We showed previously that expression of the CUL3Δ9 protein specifically in smooth muscle interferes with expression and function of endogenous Cullin‐3, resulting in vascular dysfunction, arterial stiffness and hypertension. We tested the hypothesis herein that conditional deletion of Cullin‐3 specifically in smooth muscle would cause severe hypertension. We bred mice carrying a “floxed” conditional allele of Cullin‐3 with mice expressing tamoxifen‐inducible CREERT2 under control of the smooth muscle myosin heavy chain promoter. Tamoxifen (75 mg/kg) or corn oil was administered by i.p injection to 3‐month old mice for 5 consecutive days to generate mice lacking Cullin‐3 specifically in smooth muscle (S‐CUL3KO) or littermate controls, respectively. PCR analysis of genomic DNA from S‐CUL3KO mouse tissues showed Cre‐dependent recombination in smooth muscle‐enriched tissues such as carotid artery, stomach, bladder, with little evidence of recombination in brain and liver. Molecular analysis in aorta showed that Cullin‐3 protein was undetectable whereas Cullin‐1 was abundantly expressed in S‐CUL3KO mice. We then assessed vascular function in the cerebral basilar artery and aorta using a pressurized and a wire myograph, respectively, and measured blood pressure (BP) by radiotelemetry. S‐CUL3KO mice exhibited significantly increased systolic BP (SBP) at 2 weeks and 4 weeks post tamoxifen administration compared to control mice (2 weeks SBP (mm Hg): 145±1 S‐CUL3KO vs 115±2 control, p<0.001; 4 weeks SBP: 169±1 S‐CUL3KO vs 115±3 control, p<0.001). Aorta from S‐CUL3KO mice exhibited significantly impaired vasorelaxation to acetylcholine (ACh) (at 100 μM: 1.0±3% S‐CUL3KO vs 77±5% control, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 15±4% S‐CUL3KO vs 96±1% control, p<0.001). Consistent with data from aorta, cerebral basilar artery from S‐CUL3KO mice also exhibited severe impairment to ACh‐ and SNP‐mediated vasorelaxation compared to control mice. An index of aortic stiffness, pulse wave velocity, was significantly increased in S‐CUL3KO mice (3.7±0.1 m/s S‐CUL3KO vs 2.2±0.1 m/s control, P<0.001). Total RhoA, a known Cullin‐3 substrate, was significantly increased in adventitia‐free media aorta from S‐CUL3KO mice (1.8±0.1 S‐CUL3KO vs 1.0±0.1 control, P<0.001). Pre‐incubation with the Rho‐kinase inhibitor (Y27632, 2 μM) significantly improved endothelium‐dependent relaxation in basilar artery and aorta of S‐CUL3KO mice. Interestingly, captopril administration (120 mg/kg/day) in drinking water for 5 days normalized BP in hypertensive S‐CUL3KO mice to baseline pre‐tamoxifen levels (SBP captopril: 116±1 mm Hg S‐CUL3KO) suggesting a role for the renin‐angiotensin system. We conclude that smooth muscle Cullin‐3 is a major determinant of vascular tone and BP regulation, and identifying novel Cullin‐3 substrates in smooth muscle would be beneficial in developing new treatments for hypertension.

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Grants from NIH and AHA