Genetic Deletion of ß-Arrestin 2 From the Subfornical Organ and Other Periventricular Nuclei in the Brain Alters Fluid Homeostasis and Blood Pressure.

BACKGROUND: ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the AT1R [angiotensin type 1 receptor])-mediated AT1R in the subfornical organ. Recently, we demonstrated that ARRB2 (ß-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical AT1R signaling. METHODS: Male and female Arrb2FLOX mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (Arrb2ICV-Cre). Arrb2FLOX mice receiving ICV-AAV-GFP were used as control (Arrb2ICV-Control). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl. RESULTS: Arrb2ICV-Cre mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, Arrb2ICV-Cre mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, Arrb2ICV-Cre mice exhibited elevated saline intake. CONCLUSIONS: Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical AT1R responses to both exogenous and endogenous ANG. Stimulation of the AT1R/ARRB axis in the brain may represent a novel strategy to treat hypertension.

Krüppel-like factor 4 in transcriptional control of the three unique isoforms of Agouti-related peptide in mice.

Agouti-related peptide (AgRP/Agrp) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp-expressing cells (Klf4Agrp-KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C, ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp. We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity.NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

Genetic Ablation of Prorenin Receptor in the Rostral Ventrolateral Medulla Influences Blood Pressure and Hydromineral Balance in Deoxycorticosterone-Salt Hypertension.

Non-enzymatic activation of renin via its interaction with prorenin receptor (PRR) has been proposed as a key mechanism of local renin-angiotensin system (RAS) activation. The presence of renin and angiotensinogen has been reported in the rostral ventrolateral medulla (RVLM). Overactivation of bulbospinal neurons in the RVLM is linked to hypertension (HTN). Previous studies have shown that the brain RAS plays a role in the pathogenesis of the deoxycorticosterone (DOCA)-salt HTN model. Thus, we hypothesized that PRR in the RVLM is involved in the local activation of the RAS, facilitating the development of DOCA-salt HTN. Selective PRR ablation targeting the RVLM (PRRRVLM-Null mice) resulted in an unexpected sex-dependent and biphasic phenotype in DOCA-salt HTN. That is, PRRRVLM-Null females (but not males) exhibited a significant delay in achieving maximal pressor responses during the initial stage of DOCA-salt HTN. Female PRRRVLM-Null subsequently showed exacerbated DOCA-salt-induced pressor responses during the "maintenance" phase with a maximal peak at 13 d on DOCA-salt. This exacerbated response was associated with an increased sympathetic drive to the resistance arterioles and the kidney, exacerbated fluid and sodium intake and output in response to DOCA-salt, and induced mobilization of fluids from the intracellular to extracellular space concomitant with elevated vasopressin. Ablation of PRR suppressed genes involved in RAS activation and catecholamine synthesis in the RVLM but also induced expression of genes involved in inflammatory responses. This study illustrates complex and sex-dependent roles of PRR in the neural control of BP and hydromineral balance through autonomic and neuroendocrine systems. Graphical abstract.

Cardiometabolic Effects of DOCA-Salt in Mice Depend on Ambient Temperature.

BACKGROUND: Mice prefer warmer environments than humans. For this reason, behavioral and physiological thermoregulatory responses are engaged by mice in response to a standard room temperature of 22 to 24 °C. Autonomic mechanisms mediating thermoregulatory responses overlap with mechanisms activated in hypertension, and, therefore, we hypothesized that housing at thermoneutral temperatures (TNs; 30 °C) would modify the cardiometabolic effects of deoxycorticosterone acetate (DOCA)-salt in mice. METHODS: The effects of DOCA-salt treatment upon ingestive behaviors, energy expenditure, blood pressure, heart rate (HR), and core temperature were assessed in C57BL/6J mice housed at room temperature or TN. RESULTS: Housing at TN reduced food intake, energy expenditure, blood pressure, and HR and attenuated HR responses to acute autonomic blockade by chlorisondamine. At room temperature, DOCA-salt caused expected increases in fluid intake, sodium retention in osmotically inactive pools, blood pressure, core temperature, and also caused expected decreases in fat-free mass, total body water, and HR. At TN, the effects of DOCA-salt upon fluid intake, fat gains, hydration, and core temperature were exaggerated, but effects on energy expenditure and HR were blunted. Effects of DOCA-salt upon blood pressure were similar for 3 weeks and exaggerated by TN housing in the fourth week. CONCLUSIONS: Ambient temperature robustly influences behavioral and physiological functions in mice, including metabolic and cardiovascular phenotype development in response to DOCA-salt treatment. Studying cardiometabolic responses of mice at optimal ambient temperatures promises to improve the translational relevance of rodent models.

ARRB2 (ß-Arrestin-2) Deficiency Alters Fluid Homeostasis and Blood Pressure Regulation.

BACKGROUND: GPCRs (G protein-coupled receptors) are implicated in blood pressure (BP) and fluid intake regulation. There is a developing concept that these effects are mediated by both canonical G protein signaling and noncanonical ß-arrestin mediated signaling, but the contributions of each remain largely unexplored. Here, we hypothesized that ß-arrestin contributes to fluid homeostasis and blood pressure (BP) regulation in deoxycorticosterone acetate (DOCA) salt hypertension, a prototypical model of salt-sensitive hypertension. METHODS: Global ß-arrestin1 (Arrb1) and ß-arrestin2 (Arrb2) knockout mice were employed to evaluate drinking behavior, and BP was evaluated in Arrb2-knockout mice. Age- and sex-matched C57BL/6 mice served as controls. We measured intake of water and different sodium chloride solutions and BP employing a 2-bottle choice paradigm with and without DOCA. RESULTS: Without DOCA (baseline), Arrb2-knockout mice exhibited a significant elevation in saline intake with no change in water intake. With DOCA treatment, Arrb2-knockout mice exhibited a significant increase in both saline and water intake. Although Arrb2-knockout mice exhibited hypernatremia at baseline conditions, we did not find significant changes in total body sodium stores or sodium palatability. In a separate cohort, BP was measured via telemetry in Arrb2-knockout and C57BL/6 mice with and without DOCA. Arrb2-knockout did not exhibit significant differences in BP before DOCA treatment when provided water alone, or when provided a choice of water and saline. However, Arrb2-knockout exhibited an increased pressor response to DOCA-salt. CONCLUSIONS: These findings suggest that in salt-sensitive hypertension, ARRB2, but not ARRB1 (ß-arrestin 1), might counterbalance the canonical signaling of GPCRs.

Cardiometabolic Consequences of Deleting the Regulator of G protein Signaling-2 (Rgs2) From Cells Expressing Agouti-Related Peptide or the ANG (Angiotensin) II Type 1A Receptor in Mice.

BACKGROUND: RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in the RGS2 gene in humans have been linked to hypertension, preeclampsia, and anxiety disorders. Mice deficient for Rgs2 (Rgs2Null) exhibit hypertension, anxiety, and altered adipose development and function. METHODS: To study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for the Rgs2 gene (Rgs2Flox) was developed. These mice were bred with mice expressing Cre-recombinase via the Agouti-related peptide locus (Agrp-Cre) to cause deletion of Rgs2 from all cells expressing Agrp (Rgs2Agrp-KO), or a novel transgenic mouse expressing Cre-recombinase via the ANG (angiotensin) type 1A receptor (Agtr1a/ AT1A) promoter encoded in a bacterial artificial chromosome (BAC-AT1A-Cre) to delete Rgs2 in all Agtr1a-expressing cells (Rgs2AT1A-KO). RESULTS: Whereas Rgs2Flox, Rgs2Agrp-KO, and BAC-AT1A-Cre mice exhibited normal growth and survival, Rgs2AT1A-KO exhibited pre-weaning lethality. Relative to littermates, Rgs2Agrp-KO exhibited reduced fat gains when maintained on a high fat diet, associated with increased energy expenditure. Similarly, surviving adult Rgs2AT1A-KO mice also exhibited increased energy expenditure. Surprisingly, given the hypertensive phenotype previously reported for Rgs2Null mice and evidence supporting a role for RGS2 in terminating AT1A signaling in various cell types, Rgs2AT1A-KO mice exhibited normal blood pressure, ingestive behaviors, and renal functions, both before and after chronic infusion of ANG (490 ng/kg/min, sc). CONCLUSIONS: These results demonstrate the development of a novel mouse with conditional expression of Rgs2 and illustrate the role of Rgs2 within selected cell types for cardiometabolic control.

Chronic intracerebroventricular infusion of angiotensin II causes dose- and sex-dependent effects on intake behaviors and energy homeostasis in C57BL/6J mice.

The renin-angiotensin system (RAS) within the brain is implicated in the control of fluid and electrolyte balance, autonomic functions, blood pressure, and energy expenditure. Mouse models are increasingly used to explore these mechanisms; however, sex and dose dependencies of effects elicited by chronic intracerebroventricular (ICV) angiotensin II (ANG II) infusion have not been carefully established in this species. To examine the interactions among sex, body mass, and ICV ANG II on ingestive behaviors and energy balance, young adult C57BL/6J mice of both sexes were studied in a multiplexed metabolic phenotyping system (Promethion) during chronic infusion of ANG II (0, 5, 20, or 50 ng/h). At these infusion rates, ANG II caused accelerating dose-dependent increases in drinking and total energy expenditure in male mice, but female mice exhibited a complex biphasic response with maximum responses at 5 ng/h. Body mass differences did not account for sex-dependent differences in drinking behavior or total energy expenditure. In contrast, resting metabolic rate was similarly increased by ICV ANG II in a dose-dependent manner in both sexes after correction for body mass. We conclude that chronic ICV ANG II stimulates water intake, resting, and total energy expenditure in male C57BL/6J mice following straightforward accelerating dose-dependent kinetics, but female C57BL/6J mice exhibit complex biphasic responses to ICV ANG II. Furthermore, control of resting metabolic rate by ANG II is dissociable from mechanisms controlling fluid intake and total energy expenditure. Future studies of the sex dependency of ANG II within the brain of mice must be designed to carefully consider the biphasic responses that occur in females.

Endothelial Cullin3 Mutation Impairs Nitric Oxide-Mediated Vasodilation and Promotes Salt-Induced Hypertension.

Human hypertension caused by in-frame deletion of CULLIN3 exon-9 (Cul3∆9) is driven by renal and vascular mechanisms. We bred conditionally activatable Cul3∆9 transgenic mice with tamoxifen-inducible Tie2-CREERT2 mice to test the importance of endothelial Cul3. The resultant mice (E-Cul3∆9) trended towards elevated nighttime blood pressure (BP) correlated with increased nighttime activity, but displayed no difference in daytime BP or activity. Male and female E-Cul3∆9 mice together exhibited a decline in endothelial-dependent relaxation in carotid artery. Male but not female E-Cul3∆9 mice displayed severe endothelial dysfunction in cerebral basilar artery. There was no impairment in mesenteric artery and no difference in smooth muscle function, suggesting the effects of Cul3∆9 are arterial bed-specific and sex-dependent. Expression of Cul3∆9 in primary mouse aortic endothelial cells decreased endogenous Cul3 protein, phosphorylated (S1177) endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Cul3∆9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid, but not by a PP1 inhibitor tautomycetin. Because NO deficiency contributes to salt-induced hypertension, we tested the salt-sensitivity of E-Cul3∆9 mice. While both male and female E-Cul3∆9 mice developed salt-induced hypertension and renal injury, the pressor effect of salt was greater in female mutants. The increased salt-sensitivity in female E-Cul3∆9 mice was associated with decreased renovascular relaxation and impaired natriuresis in response to a sodium load. Thus, CUL3 mutations in the endothelium may contribute to human hypertension in part through decreased endothelial NO bioavailability, renovascular dysfunction, and increased salt-sensitivity of BP.

Melanocortin MC4R receptor is required for energy expenditure but not blood pressure effects of angiotensin II within the mouse brain.

The brain renin-angiotensin system (RAS) is implicated in control of blood pressure (BP), fluid intake, and energy expenditure (EE). Angiotensin II (ANG II) within the arcuate nucleus of the hypothalamus contributes to control of resting metabolic rate (RMR) and thereby EE through its actions on Agouti-related peptide (AgRP) neurons, which also contribute to EE control by leptin. First, we determined that although leptin stimulates EE in control littermates, mice with transgenic activation of the brain RAS (sRA) exhibit increased EE and leptin has no additive effect to exaggerate EE in these mice. These findings led us to hypothesize that leptin and ANG II in the brain stimulate EE through a shared mechanism. Because AgRP signaling to the melanocortin MC4R receptor contributes to the metabolic effects of leptin, we performed a series of studies examining RMR, fluid intake, and BP responses to ANG II in mice rendered deficient for expression of MC4R via a transcriptional block (Mc4r-TB). These mice were resistant to stimulation of RMR in response to activation of the endogenous brain RAS via chronic deoxycorticosterone acetate (DOCA)-salt treatment, whereas fluid and electrolyte effects remained intact. These mice were also resistant to stimulation of RMR via acute intracerebroventricular (ICV) injection of ANG II, whereas BP responses to ICV ANG II remained intact. Collectively, these data demonstrate that the effects of ANG II within the brain to control RMR and EE are dependent on MC4R signaling, whereas fluid homeostasis and BP responses are independent of MC4R signaling.

RhoBTB1 reverses established arterial stiffness in angiotensin II-induced hypertension by promoting actin depolymerization.

Arterial stiffness predicts cardiovascular disease and all-cause mortality, but its treatment remains challenging. Mice treated with angiotensin II (Ang II) develop hypertension, arterial stiffness, vascular dysfunction, and a downregulation of Rho-related BTB domain-containing protein 1 (RhoBTB1) in the vasculature. RhoBTB1 is associated with blood pressure regulation, but its function is poorly understood. We tested the hypothesis that restoring RhoBTB1 can attenuate arterial stiffness, hypertension, and vascular dysfunction in Ang II-treated mice. Genetic complementation of RhoBTB1 in the vasculature was achieved using mice expressing a tamoxifen-inducible, smooth muscle-specific RhoBTB1 transgene. RhoBTB1 restoration efficiently and rapidly alleviated arterial stiffness but not hypertension or vascular dysfunction. Mechanistic studies revealed that RhoBTB1 had no substantial effect on several classical arterial stiffness contributors, such as collagen deposition, elastin content, and vascular smooth muscle remodeling. Instead, Ang II increased actin polymerization in the aorta, which was reversed by RhoBTB1. Changes in the levels of 2 regulators of actin polymerization, cofilin and vasodilator-stimulated phosphoprotein, in response to RhoBTB1 were consistent with an actin depolymerization mechanism. Our study reveals an important function of RhoBTB1, demonstrates its vital role in antagonizing established arterial stiffness, and further supports a functional and mechanistic separation among hypertension, vascular dysfunction, and arterial stiffness.

Cardiometabolic effects of DOCA-salt in male C57BL/6J mice are variably dependent on sodium and nonsodium components of diet.

Hypertension characterized by low circulating renin activity accounts for roughly 25%-30% of primary hypertension in humans and can be modeled experimentally via deoxycorticosterone acetate (DOCA)-salt treatment. In this model, phenotypes develop in progressive phases, although the timelines and relative contributions of various mechanisms to phenotype development can be distinct between laboratories. To explore interactions among environmental influences such as diet formulation and dietary sodium (Na) content on phenotype development in the DOCA-salt paradigm, we examined an array of cardiometabolic endpoints in young adult male C57BL/6J mice during sham or DOCA-salt treatments when mice were maintained on several common, commercially available laboratory rodent "chow" diets including PicoLab 5L0D (0.39% Na), Envigo 7913 (0.31% Na), Envigo 2920x (0.15% Na), or a customized version of Envigo 2920x (0.4% Na). Energy balance (weight gain, food intake, digestive efficiency, and energy efficiency), fluid and electrolyte homeostasis (fluid intake, Na intake, fecal Na content, hydration, and fluid compartmentalization), renal functions (urine production rate, glomerular filtration rate, urine Na excretion, renal expression of renin, vasopressin receptors, aquaporin-2 and relationships among markers of vasopressin release, aquaporin-2 shedding, and urine osmolality), and blood pressure, all exhibited changes that were subject to interactions between diet and DOCA-salt. Interestingly, some of these phenotypes, including blood pressure and hydration, were dependent on nonsodium dietary components, as Na-matched diets resulted in distinct phenotype development. These findings provide a broad and robust illustration of an environment × treatment interaction that impacts the use and interpretation of a common rodent model of low-renin hypertension.