Rgs5 targeting leads to chronic low blood pressure and a lean body habitus

Abstract

RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

FIG. 1.

Generation of Rgs5-targeted mice. (A) Schematic of Rgs5 targeting construct to generate RGS5-deficient mice. Parts of exon 1 and intron 1 of the mouse Rgs5 gene were replaced with a cassette comprising the lacZ gene and a neomycin resistance marker (Neo). Arrows indicate the direction of transcription. Restriction enzymes: A, AvrII; H, HindIII; P, PacI; R, EcoRI; RV, EcoRV; and S, SmaI. (B) PCR analysis of genomic DNA from Rgs5^+/+ (+/+), Rgs5^+/− (+/−), and Rgs5^−/− (−/−) mice to verify Rgs5 gene targeting. The PCR product from wild-type genomic DNA is 1.6 kb, while the product generated from RGS5-deficient mice is 1.5 kb. (C) RT-PCR analysis to assess the expression of Rgs2, Rgs4, Rgs5, Rgs16, Gnai1 (Gi1), Gnai2 (Gi2), Gnai3 (Gi3), Gnaq (Gq), and Gnas (Gs) mRNAs. PCR using gene-specific primers was performed with cDNA generated from aVSMC and aorta tissue. Actin primers were used to monitor cDNA synthesis. Two to three independent experiments were performed and showed similar results.

FIG. 2.

RGS5-deficient mice are lean. (A) Body weights of male (M) and female (F) Rgs5^+/+ and Rgs5^−/− mice were measured at 2 (2 M), 10 (10 M), and 15 (15 M) months. The numbers of animals (n) used are shown. Data shown are the means ± standard errors of the means (SEM). Both male and female homozygous mice had significantly reduced body weights compared to those of age-matched littermate controls. (B) Female Rgs5^+/+ and Rgs5^−/− mice aged 7 and 1/2 months old were photographed. (C) CT was performed with 10-month-old mice injected with Fenestra-VC. Relative fat contents were obtained by the analysis of reconstructed three-dimensional images using Amira 4.1. The numbers of animals used are shown. Data shown are the means ± SEM. Data were analyzed for statistical significance by Student's t test, and P values are indicated above the bars.

FIG. 3.

RGS5-deficient mice are hypotensive. Three-day means of arterial pressure measurements for Rgs5^+/+ and Rgs5^−/− mice are shown. For each genotype, five male and three female mice were used. Mice were acclimated to the BP machine for 2 days, and the actual BPs were measured 20 times consecutively after 10 cycles of mock measurement for 3 days. Acclimation and measurement were performed in the morning with nonanesthetized mice. Data shown are the means ± SEM. Data were analyzed for statistical significance by Student's t test.

FIG. 4.

The aortas in RGS5-deficient mice are dilated. (A) Representative images of VGE-stained aorta sections from Rgs5^+/+ and Rgs5^−/− mice are shown. Aortas were harvested from four mice of each genotype, and sections were made from three areas: ascending (proximal to the heart), midthoracic, and descending (proximal to the diaphragm). (B and C) Morphometric analysis of aortas. The mean inner and outer circumferences (B) and mean relative medial thicknesses (medial area/total area × 100) (C) of sections from four mice of each genotype are shown. Numbers above the bars in panel B are P values. (D) Representative images of VGE-stained kidney resistance vessels (diameters of 50 to 100 μm) from Rgs5^+/+ and Rgs5^−/− mice. (E) Morphometric analysis of the renovasculature. The mean relative medial thicknesses of approximately 20 kidney resistance vessels from each genotype are shown. Data are the means ± SEM. Data were analyzed for statistical significance by Student's t test. Images were captured with Image-Pro, and data were obtained by using the measurement module of Image-Pro.

FIG. 5.

Signaling assays. Aortic smooth muscle cells were isolated from Rgs5^+/+ and Rgs5^−/− mice and cultured in Dulbecco's modified Eagle's medium-F12 containing 20% fetal calf serum. Cells were serum starved for 24 h and treated with angiotensin II (AngII; 1 μM), ET-1 (100 nM), isoproterenol (ISO; 10 μM), S1P (100 nM), or SNP (1.5 mM) for the times indicated (in minutes). Cells were then lysed in a lysis buffer and used for Western blot analysis with anti-phospho-ERK antibody (pERK) and antibodies to phospho-VASP phosphorylated at Ser157 [pVASP(157)] and Ser239 [pVASP(239)]. The same protein membranes were immunoblotted with antiactin antibody for the normalization of protein loading. Two to five independent experiments were performed and showed similar results. WT, wild type; KO, knockout; −, unstimulated.