Genetic Deletion of β-Arrestin-2 from the Subfornical Organ and other Periventricular Nuclei in the Brain Alters Fluid Homeostasis and Blood Pressure

Abstract

Background:

Angiotensin II (ANG) elicits dipsogenic and pressor responses via activation of the canonical Gαq-mediated angiotensin type 1 receptor (AT₁R) in the subfornical organ (SFO). Recently, we demonstrated that β-arrestin2 (Arrb2/ARRB2) 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 SFO, counterbalances maladaptive dipsogenic and pressor responses to the canonical AT₁R signaling.

Methods:

Male and female Arrb2^FLOX mice received intracerebroventricular (ICV) injection of either adeno-associated virus (AAV)-Cre-GFP to induce brain-specific deletion of ARRB2 (Arrb2^ICV-Cre). Arrb2^FLOX mice receiving ICV-AAV-GFP were used as control (Arrb2^ICV-Control). Infection with ICV AAV-Cre primarily targeted the SFO with few off-targets. Fluid intake was evaluated using the two-bottle choice paradigm with one bottle containing water and one containing 0.15 mol/L NaCl.

Results:

Arrb2^ICV-Cre mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, Arrb2^ICV-Cre mice exhibited a significant increase in saline intake compared to 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, Arrb2^ICV-Cre mice exhibited elevated saline intake.

Conclusions:

Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the SFO, counterbalances canonical AT₁R responses to both exogenous and endogenous ANG. Stimulation of the AT₁R/β-arrestin axis in the brain may represent a novel strategy to treat hypertension.

Graphical Abstract

Introduction

The renin-angiotensin system (RAS) is a critical regulator of blood pressure, metabolic function, fluid intake and electrolyte balance. Angiotensin II (ANG) is the primary product of the RAS, exerting a majority of its physiological effects through the ANG type 1 receptor (AT₁R).¹ It is generally accepted that overactivation of the RAS is one of the causative factors of hypertension (HTN) as evidenced by the effectiveness of AT₁R blockers as first-line therapy to treat this condition.²

In addition to the circulating RAS, a paracrine/autocrine and tissue-specific RAS has also been described. Bickerton and Buckley first showed that central ANG induced a potent increase in blood pressure leading to the emergence of the concept of a RAS within the brain.³ Then, Booth and others showed that central ANG induced a potent dipsogenic response.^4,5 Since then, hundreds of studies have clearly demonstrated that ANG has actions in the central nervous system that are involved in autonomic regulation to the heart, kidney and vasculature which control BP, and regulatory function which affect metabolism, sodium intake, and cognitive function.^6–10

Within the brain, the actions of ANG are dictated by the neural circuits involved. The subfornical organ (SFO), organum vasculosum of the terminalis (OVLT), area postrema and median preoptic nucleus (MnPO) are well studied circumventricular organs (CVOs) – defined as regions lacking the blood brain barrier. Proper functioning of the CVOs are in part responsible for the physiological effects of circulating ANG as well as contribution to central mechanisms of brain-derived ANG.^11–13 Detection of ANG by the CVOs initiates a synchronized homeostatic response, including a variety of physiological mechanisms to induce fluid ingestion, sympathetic nervous system activation and hormonal release from the pituitary. This response involves not only behavioral responses, but autonomic and neuroendocrine functions as well. Therefore, an interplay of all these angiotensinergic pathways in the brain greatly contributes to BP regulation.

In particular, the SFO has been proposed to be the initial site of action responsible for this response.¹⁴ Studies focusing on the SFO demonstrated that specific injections of ANG into this region exerts an increase in water and sodium intake, as well as an increase in BP.^11,15 Likewise, genetic deletion of AT₁R or lesions to the SFO were demonstrated to blunt drinking behavior and pressor responses to different experimental HTN models including deoxycorticosterone acetate (DOCA)-salt, ANG-infusion and 2-kidney 1-clip.^15–17 These studies suggest the pivotal role of CVOs in ANG-mediated signaling, specifically the SFO, as it has shown high sensitivity for ANG levels in the brain.¹⁸

It is well accepted that most of the physiological actions of ANG are mediated through the G-protein component of the AT₁R (Gαq). Like most G protein coupled receptors (GPCR), AT₁R has multiple mechanisms to dampen the G protein response. For example, β-arrestin-mediated action downstream of AT₁R activation terminates Gαq activation leading to receptor internalization and signal desensitization.^19,20 There is also evidence supporting a non-canonical signaling role for β-arrestin downstream of AT₁R.^21–23 Indeed, activation of this non-canonical pathway has gained a lot of attention as it has shown to counterbalance maladaptive G protein signaling during disease.²⁴ Our prior work showed that central activation of the AT₁R β-arrestin axis using the biased ligand TRV120027 (TRV027) lowered BP and reduced 0.15 mol/L saline intake during DOCA-salt treatment.²⁵ Further, we demonstrated that global genetic deletion of β-arrestin-2 (Arrb2/ARRB2) resulted in higher saline intake and an exacerbated pressor response to DOCA-salt treatment.²⁶ These studies suggest that the beneficial effects of central TRV027 may be mediated by ARRB2. However, the involvement of ARRB2 within specific brain regions and whether they contribute to BP and sodium ingestive behaviors remains unclear. In the present study, we investigate the role of ARRB2 in BP regulation and sodium intake in mice carrying brain-specific deletion of ARRB2.

Methods

The authors declare that all supporting data are available within the article and its online supplementary files. If readers wish to see other data, they are available from the corresponding author upon reasonable request.

Procedures for animal breeding, two-bottle experimental design, water- and salt-intake measurements, urine and plasma analyses, body composition, and BP are described in the accompanying Online Supplemental Methods.^25–29

Animals:

All experiments were conducted in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and were approved by the Medical College of Wisconsin Animal Care and Use Committee. This study was conducted in C57BL/6J (Stock 000664), β-Arrestin1-deficient (Arrb1-KO; Stock 011131), and β-Arrestin2-deficient (Arrb2-KO; Stock 011130) mice as we reported previously, and Ai9 Cre-inducible TdTomato (Stock #:007909) mice from the Jackson Laboratories (Bar Harbor, ME).²⁶ Mice carrying exon2-floxed β-Arrestin2 (Arrb2^flox/flox) were kindly provided by Dr. Howard A. Rockman, Duke University.²⁷

Statistics:

All data are presented as mean ± standard error of the mean (SEM). Parametric analyses were used throughout, including 2-way and 3-way ANOVA with or without repeated measures and followed by selected (Sidak) or all pairwise (Tukey) multiple comparison procedures or independent t-test. P<0.05 was considered statistically significant.

Results

Global Deletion of β-arrestin-2 Results in Higher Pressor Response to ICV-ANG

To evaluate the role of β-arrestin in BP responses to ICV ANG, global Arrb1-KO, Arrb2-KO, and C57BL/6 control mice were subjected to acute BP recording upon ICV-ANG infusion. One week after implantation of an ICV cannula and radiotelemetric transducer, BP was recorded in conscious and unrestrained mice during the light phase (10:00 – 14:00). After BP stabilization, ANG was infused (0.2 μg/min, ICV, for 5 min to deliver a total of 1 μg) and BP was continually recorded for 30 minutes post infusion (Figure 1A). At baseline, Arrb2-KO mice exhibited higher systolic BP (SBP) and no difference in heart rate (HR) when compared to C57BL/6 and Arrb1-KO mice (Figure S1). BP tracings were then analyzed to calculate the change in systolic, mean, diastolic BP (ΔSBP, ΔMBP, ΔDBP), and ΔHR from baseline. ICV ANG caused an increase in SBP, DBP and MAP from baseline in all groups irrespective of genotype (Figure 1B1; Figure S1A, Figure S2). This increase in BP occurred concomitantly with a decrease in HR (Figure 1C1; Figure S1B). Arrb2-KO mice showed significantly higher SBP at the time of the group peak response (10-minute value) when compared to Arrb1-KO and C57BL/6, and higher SBP at maximum response determined on an individual mouse basis (max value per animal up to 15 minutes post infusion) when compared to Arrb1-KO (Figure 1B2). As measured by the area under the curve (AUC; t=1–15 min), Arrb2-KO mice exhibited a significantly higher SBP per time when compared to C57BL/6 and Arrb1-KO mice (Figure 1B3). Arrb2-KO mice also showed a faster increase in SBP as depicted by the higher slope to peak response (Figure S3). ICV ANG induced similar levels of bradycardia in Arrb1-KO and Arrb2-KO, which was more potent than C57BL/6 (Figure 1C2; Figure 1C3; Figure S1B). No significant sex differences (Sex x Genotype X ANG) were found in the pressor or bradycardic responses to ICV ANG (Figure S4).

Figure 1. Global β-Arrestin-2-Deficiency Exacerbates the Pressor Response to ICV-ANG.

A) Schematic representation of acute stimulation of BP by intracerebroventricular (ICV) infusion of ANG (1μg infused at 0.2 μg/min over 5 min). B1 and C1) ΔSBP and ΔHR over 30 min following ICV Ang II. B2 and C2) Individual ΔSBP and ΔHR values at peak response (10 min value) and max response (max value per mouse t=1–15 min). B3 and C3) Area under the curve (AUC) for SBP and HR (t=0–15 min). Data are expressed as mean±SEM. One-way ANOVA with post-hoc Tukey’s test was performed. *P < 0.05 Arrb2-KO vs C57BL/6, ^#P < 0.05 Arrb2-KO vs Arrb1-KO, ^{^}P < 0.05 Arrb1-KO vs C57BL/6. C57BL/6 (n=14), Arrb1-KO (n=12) and Arrb2-KO (n=18).

Ablation of β-arrestin-2 From Cardiovascular Brain Nuclei

Because these effects on blood pressure were much more pronounced in Arrb2 vs Arrb1 mice, we focused the remaining studies on Arrb2. Homozygous mice carrying a conditional allele of the Arrb2 gene (Arrb2^Flox) were employed, and brain-specific deletions were induced by ICV (lateral ventricle) microinjection of adeno associated virus (AAV) encoding Cre-recombinase and a fluorescent protein. As a first validation step, genomic DNA was isolated from the liver. Injection of AAV-Cre-mCherry induced recombination and deletion of Arrb2 exon 2 in liver tissue as evidenced by the presence of the PCR band at 560bp (Figure S5A).

Next, ICV injection of AAV-Cre-GFP was performed to induce deletion of Arrb2 in cardiovascular nuclei accessible to the lateral ventricle. We and others have reported that lateral ventricle injection of adenovirus and AAV efficiently infects the subfornical organ (SFO), and other sites to much lesser degree.^17,30–32 As a second validation step, double transgenic mice carrying Arrb2^Flox and Cre-activable tdTomato reporter gene (Arrb2^Flox x Ai9) were subjected to ICV-stereotactic microinjections of AAV-Cre-GFP or control virus AAV-GFP (Figure 2A). ICV injection of AAV-Cre-GFP induced a robust tdTomato signal in the SFO and a smaller subset of neurons from other brain nuclei including the median preoptic nucleus (MnPO), paraventricular nucleus (PVN) and arcuate nucleus (ARC) (Figure 2B). Analysis of whole brain slices confirmed that AAV infection was restricted to the nuclei listed above and ependymal cells surrounding the ventricle, with little to no infection elsewhere in the brain parenchyma (Figure S6). Mice subjected to ICV AAV-Cre exhibited decreased level of Arrb2 mRNA from SFO punches but not from cortex punches (Figure S5B). Of note, given the size constraint of the SFO, punches likely contain tissue that is not SFO which was not infected with AAV-Cre, and thus, the decrease in Arrb2 mRNA in the SFO might be markedly underestimated. Finally, to validate the ablation of Arrb2 in AT₁R positive neurons, double transgenic mice carrying a Cre-activable TdTomato reporter and artificial chromosome containing GFP under the control of the Agtr1a promoter (NZ44 x Ai9) were subjected to ICV microinjections of AAV-Cre (Figure 2C).^28,29,33 In these mice, red and green fluorescence was detected in the SFO and other peri-ventricular nuclei (Figure 2D). There was extensive co-localization of red and green fluorescence in the SFO (quantified in Figure S5C). There were very few co-stained neurons in the MnPO, and few if any co-labeled neurons in the PVN or ARC. Thus, from a perspective of targeting AT₁R-postiive neurons, we can conclude that the injections were largely SFO-specific. Given the effectiveness of ICV-AAV infections to target relevant cardiovascular nuclei, all subsequent experiments were designed to evaluate the role of ARRB2 in these regions on BP and fluid intake.

Figure 2. Deletion of β-Arrestin-2 from Cardiovascular Nuclei.

A) Schematic of double transgenic mouse model carrying Arrb2^Flox x Ai9 reporter (CAG-stop^flox-tdTomato). Cre-mediated recombination causes deletion of Arrb2^Flox and induces expression of TdTomato. B) Widespread tdTomato (red) reporter activated by ICV AAV-Cre in the subfornical organ (SFO), with lesser expression in the median preoptic nucleus (MnPO), paraventricular nucleus (PVN) and arcuate nucleus (ARC). C) Schematic of double transgenic mouse model carrying NZ44 (BAC-AT_1AR-GFP) X Ai9 (CAG-stop^flox-tdTomato). Green cells act as a reporter for AT₁R-expressing cells and tdTomato expression is induced in response to Cre-recombinase. D) TdTomato (red) reporter activated by ICV AAV-Cre and eGFP (green) expressed via the NZ44 AT_1A BAC transgene in SFO, MnPO, PVN and ARC. Yellow color in these merged images denotes tdTomato expression in AT₁R-containing cells.

Ablation of β-arrestin-2 in the SFO and Periventricular Nuclei Results in Greater Pressor Responses to ICV-ANG

We first evaluated the role of Arrb2 within the targeted nuclei by measuring BP and HR responses to ICV ANG. Arrb2^Flox mice were subjected to ICV injections of AAV-GFP (Arrb2^ICV-Control) or AAV-Cre-GFP (Arrb2^ICV-Cre). Three weeks after recovery, mice were implanted with an ICV cannula and radiotelemetric transducer to record BP in conscious and unrestrained mice. After BP stabilization, ANG was infused ICV for 5 minutes and BP was recorded for 30 minutes post infusion (Figure 3A). Despite baseline SBP being indistinguishable between Arrb2^ICV-Control and Arrb2^ICV-Cre mice, the latter exhibited a significant reduction in HR (Figure S7). BP tracings were then analyzed to calculate the ΔSBP, ΔMBP, ΔDBP and ΔHR from baseline. ICV ANG caused an increase in SBP, DBP and MAP from baseline in both Arrb2^ICV-Control and Arrb2^ICV-Cre mice (Figure 3B1; Figure S7A; Figure S8). This increase in BP was concomitant with a decrease in HR (Figure 3C1). Arrb2^ICV-Cre mice showed significantly higher SBP at maximum response when compared to Arrb2^ICV-Control (Figure 3B2). As measured by the area under the curve (AUC; t=1–15 min), Arrb2^ICV-Cre mice exhibited a significantly higher SBP when compared to Arrb2^ICV-Control mice (Figure 3B3). Although ICV ANG did not induce a significant bradycardic response in Arrb2^ICV-Cre when compared to control (Figure 3C2, Figure 3C3), Arrb2^ICV-Cre exhibited a lower HR versus baseline (Figure S7B). Despite Arrb2^ICV-Cre mice exhibiting a higher slope and apparent faster pressor response, this did not reach statistical significance (Figure S9). Overall, no significant sex differences were found in the pressor or bradycardic responses to ICV ANG, except that sex modified the effect of genotype on maximal HR response (Figure S10).

Figure 3. Genetic Deletion of β-Arrestin-2 from Select Regions of the Brain Exacerbates the Pressor Response to ICV ANG.

A) Schematic representation of acute stimulation of BP by intracerebroventricular (ICV) ANG. B1 and C1) ΔSBP and ΔHR over 30 min following ICV ANG. B2 and C2) Individual ΔSBP and ΔHR values at peak response (10 min value) and max response (max value per mouse t=1–15 min). B3 and C3) Area under the curve (AUC) for SBP and HR (t=0–15 min). Data are expressed as mean±SEM. t-test was performed. *P < 0.05 Arrb2^ICV-Cre vs Arrb2^ICV-Control, ^τP < 0.05 Arrb2^ICV-Control or Arrb2^ICV-Cre vs their untreated baseline. Arrb2^Control (n=10–11) and Arrb2^ICV-Cre (n=10–11).

Ablation of β-arrestin-2 in the SFO and Periventricular Nuclei Results in Higher Saline Intake

To evaluate the role of Arrb2 signaling in the regulation of water and sodium intake, Arrb2^ICV-Control and Arrb2^ICV-Cre mice were subjected to the two-bottle choice paradigm (Figure 4A). First, mice were presented with a choice of 2 water-filled burettes. There were no differences in daily water intake or side bias (left versus right burette) between the groups, averaging ≈50% (Figure 4B). Arrb2^ICV-Control and Arrb2^ICV-Cre mice were next presented with a choice of water versus 0.15 mol/L NaCl. While there were no differences in water intake and total fluid intake, Arrb2^ICV-Cre mice exhibited a further increase in saline intake and saline preference (Figure 4C). Sex significantly modified the effect of the virus upon total fluid intake, blunting effects in control males (Figure S11A), but the effect of sex on saline preference did not reach statistical significance (Figure S11B).

Figure 4. Genetic Deletion of β-Arrestin-2 from Select Regions of the Brain Exacerbates Saline Intake.

A) Schematic representation of the two-bottle choice experimental protocol. B) Total daily water intake and side bias when mice were presented with 2 burettes each filled with water (water vs. water). C) Water intake, saline intake, total fluid intake (calculated as total water intake plus total saline intake), and saline preference (calculated as the percentage of total saline intake versus total fluid intake) when mice were presented with a choice of water and saline (water vs. 0.15 mol/L NaCl). Data are expressed as mean±SEM. Student’s t-test was performed. Arrb2^ICV-Control (n=13) and Arrb2^ICV-Cre (n=18). * P < 0.05 Arrb2^ICV-Cre vs Arrb2^ICV-Control.

We next evaluated the role of brain Arrb2 in drinking behavior under conditions that elevate endogenous ANG levels, such as water and sodium restriction.³⁴ Arrb2^ICV-Control and Arrb2^ICV-Cre mice were placed in metabolic cages and were provided standard chow and water. First, water was restricted by removing the water burettes for an overnight period. Access was restored by providing the mice a two-bottle choice of water and 0.15 mol/L NaCl. Volumes in each burette were measured for 4 hours after restoration (Figure 5A). To first validate a negative water balance, body composition and plasma were analyzed. At the end of the water restriction period Arrb2^ICV-Control and Arrb2^ICV-Cre mice exhibited a similar ~13% reduction in total body water (Figure 5B). Arrb2^ICV-Control and Arrb2^ICV-Cre mice also exhibited a modest increase in plasma osmolality, a significant increase in plasma hematocrit (Hct), and a significant increase in plasma Na concentration when compared to a control reference (REF) group consisting of Arrb2^Flox mice not treated with virus and not subjected to water restriction (Figure 5B). All these are characteristic parameters of acute dehydration. Mice were then presented with the two-bottle choice paradigm after water restriction. Both Arrb2^ICV-Control and Arrb2^ICV-Cre mice drank ~50% of the final amount of total fluid within the first 30 minutes. There was no difference in water intake at any timepoint. However, Arrb2^ICV-Cre mice exhibited an exaggerated increase in saline intake throughout the 4-hour repletion. This increase in saline intake in Arrb2^ICV-Cre mice was reflected in a significantly increased preference for saline when compared to Arrb2^ICV-Control (Figure 5C).

Figure 5. Water Restriction Augments Saline Intake in Mice with Genetic Deletion of β-Arrestin-2 from Select Regions of the Brain.

A) Schematic representation of the experimental design. B) Total body water, plasma osmolality, hematocrit, and plasma Na concentration after the water restriction period. One-way ANOVA was performed. * P < 0.05 Arrb2^ICV-Control or Arrb2^ICV-Cre vs reference group (REF). C) Cumulative water, saline, total fluid intake and preference for 0.15 mol/L during 4-hour access restoration. Repeated-measures two-way ANOVA, P values shown in each figure. Data are expressed as mean±SEM. Arrb2^ICV-Control (n=11) and Arrb2^ICV-Cre (n=11). In the 4th panel in C * P < 0.05 Arrb2^ICV-Cre vs Arrb2^ICV-Control.

In an independent cohort, sodium was depleted by administering two furosemide injections (IP) two hours apart and supplying the mice with a customized, low Na (0.04%) version of the diet plus deionized water for an overnight period. Access was restored by providing the mice a two-bottle choice of water and 0.15 mol/L NaCl. Fluid from each burette was measured for four hours after restoration (Figure 6A). At the end of the sodium depletion period, Arrb2^ICV-Control and Arrb2^ICV-Cre mice similarly exhibited a ~20% reduction in total body water (Figure 6B). They also similarly exhibited a significant increase in plasma Hct and a significant decrease in plasma Na when compared to the control reference group (same as in Figure 5). Arrb2^ICV-Cre but not Arrb2^ICV-Control mice exhibited significantly higher plasma osmolality when compared to reference group. When mice were presented to the two-bottle choice paradigm after Na restriction, Arrb2^ICV-Cre and controls markedly drank more saline when compared to water (Figure 6C, note differences in Y-axis scale). While Arrb2^ICV-Cre mice exhibited significant increase in saline intake at 1-hour, the pattern of drinking behavior was similar between the groups thereafter showing no differences in saline preference.

Figure 6. Sodium Restriction Augments Water Intake in Mice with Genetic Deletion of β-Arrestin-2 from Select Regions of the Brain.

A) Schematic representation of the experimental design. B) Total body water, plasma osmolality, hematocrit, and plasma Na concentration after the sodium depletion period. One-way ANOVA was performed, *P < 0.05 Arrb2^ICV-Control or Arrb2^ICV-Cre vs reference group (REF). C) Cumulative water, saline, total fluid intake and preference for 0.15 mol/L saline during 4-hour access restoration. Repeated-measures two-way ANOVA, P values shown in each figure. Data are expressed as mean±SEM. Arrb2^ICV-Control (n=11) and Arrb2^ICV-Cre (n=11). * P < 0.05 Arrb2^ICV-Cre vs Arrb2^ICV-Control.

Discussion

The present study provides compelling evidence implicating the role of ARRB2 in the brain in the regulation of BP, HR, and fluid intake. First, our results demonstrate that mice carrying a global deletion of ARRB2 exhibit an exaggerated pressor and bradycardic response to ICV infusion of ANG. Second, mice carrying selective deletion of ARRB2 from selective cardiovascular nuclei in the brain, particularly the SFO exhibit: 1) Normal BP but lower resting HR, 2) exacerbated pressor response to ICV infusion of ANG, 3) elevated saline intake under baseline conditions and under conditions that naturally elevate endogenous ANG levels. These results support the role of ARRB2 in brain- and ANG-dependent mechanisms involved in the regulation of BP and consumption of sodium.

Decades of studies have clearly established that ICV ANG leads to an increase in BP and central blockade of the AT₁R blunts or blocks that response.^35–39 In our previous studies we showed that global Arrb2-KO mice exhibited a further elevation in BP when subjected to DOCA-salt HTN.^25,26 This suggests that during brain RAS activation, lack of ARRB2 potentiates the pressor response to ANG. However, the use of a global KO and the DOCA-salt model lacks neuroanatomical and AT₁R specificity. To initially assess brain-specificity of the ARRB2 response, we evaluated BP to direct infusion of ANG into the brain of global Arrb-1-KO and Arrb-2 KO mice. At baseline, despite no differences in HR, Arrb2-KO mice showed higher SBP. This suggests an increase in sympathetic tone to vascular beds and thus increase in total peripheral resistance that might be attributed to continuous Gαq activation due to lack of ARRB2. It has been described that myogenic constriction of vascular smooth muscles is predominantly mediated by Gαq activation.⁴⁰ We then demonstrated that global Arrb2-KO mice exhibited an exacerbated pressor response to ICV ANG. Presumably, these data suggest that deficiency of ARRB2 either prolongs or sensitizes AT₁R-containing angiotensinergic neurons to elevate BP. Interestingly, although Arrb1-KO mice did not show further elevation in BP when compared to Arrb2-KO mice, they did show similar reduction in HR. It remains unclear whether the small elevation in the pressor response is attributed to compensatory parasympathetic-mediated mechanisms. Indeed, ANG has been localized to pre-ganglionic fibers and brain regions involved in vagal innervation to the heart.⁴¹ ANG signaling in these regions is suggested to inhibit parasympathetic signaling to the heart, as blockade of the AT₁R potentiates the bradycardic response to vagal nerve stimulation.⁴² Thus, it remains possible that ARRB2 deletion not only augments the pressor response to ICV ANG, but also suppresses the vagal response leading to exacerbated BP. However, further studies blocking the parasympathetic mechanisms are required to elucidate this.

The physiological output of angiotensinergic signaling in the brain is dependent on the location and neural circuitry engaged. We performed a targeted deletion of ARRB2 from selected nuclei by performing ICV injection of AAV-Cre in mice carrying floxed Arrb2 (Arrb2^ICV-Cre) alleles. The specificity and selectivity of the ICV viral injections were confirmed in mice carrying Cre-activatable tdTomato reporter in which we observed a strong tdTomato signal in regions surrounding the lateral and third ventricles with the highest staining in the SFO and lower staining in the MnPO, PVN and ARC. Notably, the largest percentage of tdTomato expression in AT₁R-containing neurons appeared in the SFO with much fewer numbers of co-labeled neurons in the MnPO, and very few in the PVN and ARC. A reduction in Arrb2 mRNA transcription was demonstrated in the SFO. Unfortunately, preliminary RNAscope experiments designed to distinguish Arrb1 from Arrb2 mRNA were not successful because we were unable to validate the specificity of the probes. Transcript levels were not measured in the other regions because of the challenges in precisely isolating those regions. Thus, we recognize this limitation and cannot attribute all the phenotypes to specific deletion of ARRB2 in the SFO.

We next evaluated BP to direct infusion of ICV ANG in Arrb2^ICV-Cre mice. At baseline, despite no differences in SBP, Arrb2^ICV-Cre mice exhibited lower HR. Proposing mechanism(s) that cause bradycardia in these mice with the current available data is challenging. However, we can speculate that there are two possible ways that a lack of ARRB2 in the brain can cause cardioinhibitory effects. One could be a direct effect on pre-autonomic neurons causing either lower cardiac sympathetic tone, higher parasympathetic tone, or both. Alternatively, we cannot rule out the existence of an underlying masked phenotype that is compensated by vagal cardioinhibitory reflexes. Nevertheless, several additional studies evaluating cardiac and autonomic function in great detail would be necessary to infer a possible explanation to this phenotype.

Next, consistent with the pressor response exhibited in the Arrb2-KO mice, Arrb2^ICV-Cre mice also exhibited an exacerbated pressor response to ICV infusion of ANG implying that the potentiated response to ICV ANG in the global KO mice might be mediated by lack of ARRB2 in the brain. Conceptually, this suggests that ARRB2 in the brain serves as a protective mechanism buffering states of brain RAS overactivity. This concept can be extended to other brain nuclei including autonomic centers in the brainstem as previous studies have shown that overexpression of ARRB1 or ARRB2 in the rostral ventrolateral medulla lowered BP in a genetic model of essential hypertension.^43,44 Future studies ablating either ARRB1 or ARRB2 in this region or in other regions controlling sympathetic outflow are warranted.

In addition to its role in BP control, the brain RAS also participates in the control of fluid ingestive behaviors.¹¹ In our previous studies we showed that global Arrb2-KO mice exhibited a further increase in saline and water intake when subjected to DOCA-salt HTN.^25,26 Similar to the BP effects, we speculate that lack of ARRB2 might potentiate behavioral responses to central elevations of ANG. Consequently, we subjected Arrb2^ICV-Cre mice to a two-bottle choice to evaluate water and saline intake under baseline conditions. First, we demonstrated that Arrb2^ICV-Cre exhibited higher saline intake and therefore higher preference for saline. Given the increase in saline intake of Arrb2^ICV-Cre at baseline conditions, we tested the hypothesis that conditions known to elevate endogenous brain ANG levels would exacerbate this response. Therefore, Arrb2^ICV-Cre mice were subjected to water restriction and sodium depletion protocols which disrupt the tonicity of the extracellular/intracellular spaces and elevates endogenous levels of ANG.^34,45,46

We first corroborated the efficacy of these protocols in causing dehydration. At the end of the water restriction period, Arrb2^ICV-Cre and controls exhibited overall total body water loss, leading to increased Hct, plasma Na and plasma osmolality. Similarly, at the end of the Na depletion period, Arrb2^ICV-Cre and controls exhibited overall total body water loss and increased Hct. Interestingly, despite the low Na levels, Arrb2^ICV-Cre also exhibited an increase in plasma osmolality, suggesting Arrb2^ICV-Cre might have impaired homeostatic mechanisms. Next, mice were subjected to the two-bottle choice and water and saline intake were measured hourly. Although, Arrb2^ICV-Cre and control groups consumed corresponding volumes of fluid to the amount lost (~2.0 mL total fluid intake versus ~2.5 g of water loss), the choice of fluid, either water or saline was different. After water restriction, control mice showed higher water intake compared to saline intake, which is expected to compensate for the higher Na levels. Interestingly, although no differences in water intake were observed, Arrb2^ICV-Cre showed a further increase in saline intake when compared to controls. This suggests that, albeit the elevation in Na levels, Arrb2^ICV-Cre mice continue to prefer saline implying a dysfunctional mechanism that leads to aggravation of the hypernatremic condition. Then, after Na depletion, Arrb2^ICV-Cre and control mice showed negligible water intake but higher saline intake, which again is expected to compensate for the loss of Na. However, Arrb2^ICV-Cre exhibited a significant increase in plasma osmolality, this stimulus was not strong enough to drive further water intake.

Optogenetic studies suggest that direct connections from the SFO to MnPO and OVLT primarily mediate water intake, while connections from the SFO to the BNST are known to primarily drive saline intake – respectively known as water and salt neurons.^46,47 From these studies we could speculate that the regulatory capacity of ARRB2 to buffer local RAS overactivation appears to depend on conditions that either force the suppression of salt neurons (water restriction) more than water neurons (Na depletion). By contrasting the results from the water restriction and Na depletion conditions, we hypothesize that in water restriction there is an ARRB2-mediated suppression of RAS activity within the salt circuit to BNST to compensate the hypernatremia. While in Na depletion, the hyponatremic stimulus activated the salt neurons in both Arrb2^ICV-Cre and control mice, with a further increase in Arrb2^ICV-Cre, suggesting higher sensitivity of this circuitry to levels of ARRB2. Further, the suppression of water neurons remained intact. Presumably, ANG stimulation of the salt circuitry to the BNST seem to be regulated by ARRB2 levels. However, other mechanisms involving Na-sensing channels, osmoreceptors and volume receptors cannot be ruled out.

These results complement other studies that have used direct injection, or lesioning of the SFO, demonstrating the role of the SFO not only in BP regulation to ANG but ANG-dependent drinking behavior.^{14,15,48–50} This leads us to hypothesize that the exacerbated pressor response to ICV ANG and changes in drinking behavior, specifically saline intake, are due to ARRB2 ablation in the brain, probably the SFO. It is notable that the ICV ANG-mediated pressor and dipsogenic effects, although coordinated responses, are independent from each other. This concept has been previously described, as the increase in BP occurred earlier than the increase in water intake and vasopressin levels.^15,35,49

All experiments were conducted in both males and females to account for sex as a variable. We did not observe any three-way interactions among sex and genotype and ANG suggesting that differences in drinking behavior and pressor responses were independent of sex.

We conclude that deletion of ARRB2 from the brain enhances ANG-induced pressor, bradycardic, and dipsogenic effects. This may be mediated by a combination of 1) the loss of β-arrestin-dependent termination of the Gαq signaling and therefore disinhibited G protein signaling, and 2) the loss of G protein-independent β-arrestin signaling. Limitations of this study include: 1) acute ANG interventions may not be sufficient to uncover chronic effects regarding autonomic, neuroendocrine and behavioral angiotensinergic signaling in the brain, and 2) ICV injections to ablate ARRB2 from the brain showed a stochastic distribution of recombination, suggesting that different populations of neurons might have been targeted in individual animals. Therefore, studies employing tools that would directly distinguish between AT₁R-Gαq and AT₁R-βarrestin would be necessary. Indeed, studies are currently in progress using mouse models harboring AT₁R mutants that are genetically altered to act with a G protein or β-arrestin bias.

Perspectives: The use of AT₁R-specific β-arrestin biased ligands such as TRV027 in a variety of pre-clinical disease models provides evidence that activating the AT1R β-arrestin pathway may be cardioprotective. For example, TRV027 increased cardiac contractility in rodents, whereas two different ANG receptor blockers (ARBs) had the opposite effect.⁵¹ Likewise, TRV027 lowered BP, increased cardiac output, and decreased vascular resistance in a canine model of heart failure.⁵² These studies were among a larger set of studies which provided the rationale to evaluate the effectiveness of TRV027 in humans. In the first clinical experience with TRV027, it was deemed to be safe and significantly lowered BP in patients that exhibited high plasma renin activity.⁵³ TRV027 was then tested in a multi-center, randomized, double-blind, placebo-controlled, parallel group, phase IIb dose-ranging study call the Biased Ligand of the Angiotensin II Type 1 Receptor in Patients with Acute Heart Failure (BLAST-AHF) trial.⁵⁴ Although TRV027 did not show significant clinical benefit, a post-hoc evaluation revealed that TRV027 reduced 180-day all-cause mortality and cardiovascular death or hospital readmission in patients in the two higher tertile groups for systolic BP.⁵⁵ Collectively, the results in this manuscript evidencing a beneficial role of brain β-arrestin2 in BP regulation and salt intake in combination with preclinical study and clinical trial data using TRV027 suggests that the beneficial effects of TRV027 on BP and salt intake are most likely mediated by β-arrestin2 in the brain. Thus, drugs targeting the AT₁R β-arrestin signaling pathway might represent advantageous effects over classic ARBs in patients that are refractory to current medications or those with neurogenic forms of HTN.

What is new?

• Global deletion of ARRB2 and deletion of ARRB2 from selective brain nuclei exhibited an exacerbated pressor response to ICV ANG.

• Deletion of ARRB2 from selective brain nuclei exhibited augmented saline intake at baseline and impairment of homeostatic responses after water depletion and sodium restriction.

• The role of brain ARRB2 in BP regulation and fluid homeostasis is largely sex independent.

What is relevant?

New components of the brain RAS continue to be discovered. β-arrestin activation downstream of the AT₁R has been characterized as a novel component to terminate detrimental G protein signaling during disease. Therefore, a new class of AT₁R-mediated pharmaceutical drugs that maintain the β-arrestin component might represent more efficacious therapies for patients that do not respond to current anti-hypertensives.

Clinical/Pathophysiological Implications

Clinical studies demonstrated that agonists of the AT₁R that selectively engage β-arrestin exert cardiovascular benefits in patients with high renin angiotensin activity. Further, these agonists also lower BP in heart failure patients that exhibit hypertension. Interestingly, other studies suggest that traditional AT₁R blockers fail to exert similar cardiovascular benefits of β-arrestin biased agonists. Future studies are aimed to gain further specificity in the activation of β-arrestin solely from AT₁R positive neurons.

Abbreviations:

AAV

adeno associated virus

ANG

Angiotensin II

AUC

area under the curve

ARRB1/Arrb1

β-arrestin 1

ARRB2/Arrb2

β-arrestin 2

Arrb1-KO

β-arrestin 1 knockout

Arrb2-KO

β-arrestin 2 knockout

AT₁R

angiotensin II AT1 receptor

BNST

bed nucleus of stria terminalis

BP

blood pressure

CVO

circumventricular organ

DOCA

Deoxycorticosterone acetate

GPCR

G protein coupled receptor

Hct

hematocrit

HR

heart rate

HTN

hypertension

ICV

intracerebroventricular

MnPO

median preoptic nucleus

Na

sodium

OVLT

organum vasculosum of the terminalis

PVN

paraventricular nucleus

RAS

renin-angiotensin system

SFO

subfornical organ

TRV027

TRV120027