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.

Exercise mitigates flow recirculation and activates metabolic transducer SCD1 to catalyze vascular protective metabolites.

Exercise promotes pulsatile shear stress in the arterial circulation and ameliorates cardiometabolic diseases. However, exercise-mediated metabolic transducers for vascular protection remain under-investigated. Untargeted metabolomic analysis demonstrated that wild-type mice undergoing voluntary wheel running exercise expressed increased endothelial stearoyl-CoA desaturase 1 (SCD1) that catalyzes anti-inflammatory lipid metabolites, namely, oleic (OA) and palmitoleic acids (PA), to mitigate NF-κB-mediated inflammatory responses. In silico analysis revealed that exercise augmented time-averaged wall shear stress but mitigated flow recirculation and oscillatory shear index in the lesser curvature of the mouse aortic arch. Following exercise, endothelial Scd1-deleted mice (Ldlr-/- Scd1EC-/-) on high-fat diet developed persistent VCAM1-positive endothelium in the lesser curvature and the descending aorta, whereas SCD1 overexpression via adenovirus transfection mitigated endoplasmic reticulum stress and inflammatory biomarkers. Single-cell transcriptomics of the aorta identified Scd1-positive and Vcam1-negative endothelial subclusters interacting with other candidate genes. Thus, exercise mitigates flow recirculation and activates endothelial SCD1 to catalyze OA and PA for vascular endothelial protection.

Modulatory effects of estrous cycle on ingestive behaviors and energy balance in young adult C57BL/6J mice maintained on a phytoestrogen-free diet.

The estrous cycle is known to modify food, fluid, and electrolyte intake behaviors and energy homeostasis in various species, in part through fluctuations in estrogen levels. Simultaneously, commonly commercially available rodent dietary formulations greatly vary in soy protein content, and thereby the delivery of biologically active phytoestrogens. To explore the interactions among the estrous cycle, sodium, fluid, and caloric seeking behaviors, and energy homeostasis, young adult C57BL/6J female mice were maintained on a soy protein-free 2920x diet and provided water, or a choice between water and 0.15 mol/L NaCl drink solution. Comprehensive metabolic phenotyping was performed using a multiplexed Promethion (Sable Systems International) system, and estrous stages were determined via daily vaginal cytology. When provided food and water, estrous cycling had no major modulatory effects on intake behaviors or energy balance. When provided a saline solution drink choice, significant modulatory effects of the transition from diestrus to proestrus were observed upon fluid intake patterning, locomotion, and total energy expenditure. Access to saline increased total daily sodium consumption and aspects of energy expenditure, but these effects were not modified by the estrous stage. Collectively, these results indicate that when supplied a phytoestrogen-free diet, the estrous cycle has minor modulatory effects on ingestive behaviors and energy balance in C57BL/6J mice that are sensitive to sodium supply.NEW & NOTEWORTHY When provided a phytoestrogen-free diet, the estrous cycle had very little effect on food and water intake, physical activity, or energy expenditure in C57BL/6J mice. In contrast, when provided an NaCl drink in addition to food and water, the estrous cycle was associated with changes in intake behaviors and energy expenditure. These findings highlight the complex interactions among estrous cycling, dietary formulation, and nutrient presentation upon ingestive behaviors and energy homeostasis in mice.

The 2023 Walter B. Cannon Award Lecture: Mechanisms Regulating Vascular Function and Blood Pressure by the PPARγ-RhoBTB1-CUL3 Pathway.

Human genetic and clinical trial data suggest that peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor transcription factor plays an important role in the regulation of arterial blood pressure. The examination of a series of novel animal models, coupled with transcriptomic and proteomic analysis, has revealed that PPARγ and its target genes employ diverse pathways to regulate vascular function and blood pressure. In endothelium, PPARγ target genes promote an antioxidant state, stimulating both nitric oxide (NO) synthesis and bioavailability, essential components of endothelial-smooth muscle communication. In vascular smooth muscle, PPARγ induces the expression of a number of genes that promote an antiinflammatory state and tightly control the level of cGMP, thus promoting responsiveness to endothelial-derived NO. One of the PPARγ targets in smooth muscle, Rho related BTB domain containing 1 (RhoBTB1) acts as a substrate adaptor for proteins to be ubiquitinated by the E3 ubiquitin ligase Cullin-3 and targeted for proteasomal degradation. One of these proteins, phosphodiesterase 5 (PDE5) is a target of the Cullin-3/RhoBTB1 pathway. Phosphodiesterase 5 degrades cGMP to GMP and thus regulates the smooth muscle response to NO. Moreover, expression of RhoBTB1 under condition of RhoBTB1 deficiency reverses established arterial stiffness. In conclusion, the coordinated action of PPARγ in endothelium and smooth muscle is needed to maintain NO bioavailability and activity, is an essential regulator of vasodilator/vasoconstrictor balance, and regulates blood vessel structure and stiffness.

Insights Into the Role of Angiotensin-II AT1 Receptor-Dependent ß-Arrestin Signaling in Cardiovascular Disease.

ß-arrestins are a family of intracellular signaling proteins that play a key role in regulating the activity of G protein-coupled receptors. The angiotensin-II type 1 receptor is an important G protein-coupled receptor involved in the regulation of cardiovascular function and has been implicated in the progression of cardiovascular diseases. In addition to canonical G protein signaling, G protein-coupled receptors including the angiotensin-II type 1 receptor can signal via ß-arrestin. Dysregulation of ß-arrestin signaling has been linked to several cardiovascular diseases including hypertension, atherosclerosis, and heart failure. Understanding the role of ß-arrestins in these conditions is critical to provide new therapeutic targets for the treatment of cardiovascular disease. In this review, we will discuss the beneficial and maladaptive physiological outcomes of angiotensin-II type 1 receptor-dependent ß-arrestin activation in different cardiovascular diseases.

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.

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination.

We identified Rho-related BTB domain containing 1 (RhoBTB1) as a key regulator of phosphodiesterase 5 (PDE5) activity, and through PDE5, a regulator of vascular tone. We identified the binding interface for PDE5 on RhoBTB1 by truncating full-length RhoBTB1 into its component domains. Co-immunoprecipitation analyses revealed that the C-terminal half of RhoBTB1 containing its two BTB domains and the C-terminal domain (B1B2C) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via Cullin-3 (CUL3). The C-terminal domain was essential in recruiting PDE5 as constructs lacking this region could not participate in PDE5 binding or proteasomal degradation. We also identified Pro353 and Ser363 as key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of either of these residues exhibited impaired CUL3 binding and PDE5 degradation, although the binding to PDE5 was preserved. Finally, we employed ascorbate peroxidase 2 (APEX2) proximity labeling using a B1B2C-APEX2 fusion protein as bait to capture unknown RhoBTB1 binding partners. Among several B1B2C-binding proteins identified and validated, we focused on SET domain containing 2 (SETD2). SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to pharmacological inhibition of the proteasome or Cullin complex, CUL3 deletion, and RhoBTB1-inhibition with siRNA. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies will determine whether SETD2 plays a role in cardiovascular function.

Addressing the decline in graduate students' mental well-being.

At the American Physiology Summit 2023 session entitled, "Mental Health for Graduate Students," numerous students expressed struggling with poor mental well-being primarily because of negative experiences during their graduate training. In fact, studies show that up to 50% of graduate students report symptoms of depression, anxiety, or burnout during their training, and poor mental well-being is a major contributor to students' decision to leave academia. Most of the current solutions focus on treatment or wellness strategies; while these are important and necessary, the training environment or culture that often contributes to worsening well-being continues to persist. In this collaborative article between trainees and mentors across various career stages, we discuss how the pace of scientific advancements and the associated competition, lack of sufficient support for students from diverse backgrounds, and mentor-mentee relationships crucially influence graduate students' mental well-being. We then offer specific solutions at the individual, institutional, and national levels that can serve as a starting point for improving graduate students' mental health and overall training experience.

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.

Angiotensin AT1A receptor signal switching in Agouti-related peptide neurons mediates metabolic rate adaptation during obesity.

Resting metabolic rate (RMR) adaptation occurs during obesity and is hypothesized to contribute to failed weight management. Angiotensin II (Ang-II) type 1 (AT1A) receptors in Agouti-related peptide (AgRP) neurons contribute to the integrative control of RMR, and deletion of AT1A from AgRP neurons causes RMR adaptation. Extracellular patch-clamp recordings identify distinct cellular responses of individual AgRP neurons from lean mice to Ang-II: no response, inhibition via AT1A and Gαi, or stimulation via Ang-II type 2 (AT2) receptors and Gαq. Following diet-induced obesity, a subset of Ang-II/AT1A-inhibited AgRP neurons undergo a spontaneous G-protein "signal switch," whereby AT1A stop inhibiting the cell via Gαi and instead begin stimulating the cell via Gαq. DREADD-mediated activation of Gαi, but not Gαq, in AT1A-expressing AgRP cells stimulates RMR in lean and obese mice. Thus, loss of AT1A-Gαi coupling within the AT1A-expressing AgRP neuron subtype represents a molecular mechanism contributing to RMR adaptation.

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.

Cell-specific transcriptome changes in the hypothalamic arcuate nucleus in a mouse deoxycorticosterone acetate-salt model of hypertension.

A common preclinical model of hypertension characterized by low circulating renin is the "deoxycorticosterone acetate (DOCA)-salt" model, which influences blood pressure and metabolism through mechanisms involving the angiotensin II type 1 receptor (AT1R) in the brain. More specifically, AT1R within Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) has been implicated in selected effects of DOCA-salt. In addition, microglia have been implicated in the cerebrovascular effects of DOCA-salt and angiotensin II. To characterize DOCA-salt effects upon the transcriptomes of individual cell types within the ARC, we used single-nucleus RNA sequencing (snRNAseq) to examine this region from male C57BL/6J mice that underwent sham or DOCA-salt treatment. Thirty-two unique primary cell type clusters were identified. Sub-clustering of neuropeptide-related clusters resulted in identification of three distinct AgRP subclusters. DOCA-salt treatment caused subtype-specific changes in gene expression patterns associated with AT1R and G protein signaling, neurotransmitter uptake, synapse functions, and hormone secretion. In addition, two primary cell type clusters were identified as resting versus activated microglia, and multiple distinct subtypes of activated microglia were suggested by sub-cluster analysis. While DOCA-salt had no overall effect on total microglial density within the ARC, DOCA-salt appeared to cause a redistribution of the relative abundance of activated microglia subtypes. These data provide novel insights into cell-specific molecular changes occurring within the ARC during DOCA-salt treatment, and prompt increased investigation of the physiological and pathophysiological significance of distinct subtypes of neuronal and glial cell types.

Exercise Mitigates Flow Recirculation and Activates Mechanosensitive Transcriptome to Uncover Endothelial SCD1-Catalyzed Anti-Inflammatory Metabolites.

Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ( τ ave = 50 dyne·cm -2 , ∂τ/∂t = 71 dyne·cm -2 ·s -1 , 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or τ ave) and oscillatory shear index (OSI ave ), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr -/- mice on high-fat diet but not in Ldlr -/- Scd1 EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2 , Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ( τ ave and OSI ave ) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.

Collecting duct renin regulates potassium homeostasis in mice.

AIM: The kaliuretic action of the renin-angiotensin-aldosterone system (RAAS) is well established as highlighted by hyperkalemia side effect of RAAS inhibitors but such action is usually ascribed to systemic RAAS. The present study addresses the involvement of intrarenal RAAS in K+ homeostasis with emphasis on locally generated renin within the collecting duct (CD). METHODS: Wild-type (Floxed) and CD-specific deletion of renin (CD renin KO) mice were treated for 7 days with a high K+ (HK) diet to investigate the role of CD renin in kaliuresis regulation and further define the underlying mechanism with emphasis on analysis of intrarenal aldosterone biosynthesis. RESULTS: In floxed mice, renin levels were elevated in the renal medulla and urine following a 1-week HK diet, indicating activation of the intrarenal renin. CD renin KO mice had blunted HK-induced intrarenal renin response and developed impaired kaliuresis and elevated plasma K+ level (4.45 ± 0.14 vs. 3.89 ± 0.04 mM, p < 0.01). In parallel, HK-induced intrarenal aldosterone and CYP11B2 expression along with expression of renal outer medullary K+ channel (ROMK), calcium-activated potassium channel subunit alpha-1 (α-BK), α-Na+ -K+ -ATPase, and epithelial sodium channel (ß-ENaC and cleaved-γ-ENaC) expression were all significantly blunted in CD renin KO mice in contrast to the unaltered responses of plasma aldosterone and adrenal CYP11B2. CONCLUSION: Taken together, these results support a kaliuretic action of CD renin during HK intake.

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.

Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons.

Acute regulation of CO2 and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO2/pH sensitive cells - so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO2 sensitive or insensitive based on differences in embryonic origin, suggesting these functionally distinct subpopulations may have unique transcriptional profiles. Here, we used Patch-to-Seq to determine if the CO2 responses in brainstem 5-HT neurons could be correlated to unique transcriptional profiles and/or unique molecular markers and pathways. First, firing rate changes with hypercapnic acidosis were measured in fluorescently labeled 5-HT neurons in acute brainstem slices from transgenic, Dahl SS (SSMcwi) rats expressing T2/ePet-eGFP transgene in Pet-1 expressing (serotonin) neurons (SS ePet1-eGFP rats). Subsequently, the transcriptomic and pathway profiles of CO2 sensitive and insensitive 5-HT neurons were determined and compared by single cell RNA (scRNAseq) and bioinformatic analyses. Low baseline firing rates were a distinguishing feature of CO2 sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO2 sensitive and insensitive 5-HT neurons. Pathway analyses yielded novel predicted upstream regulators, including the transcription factor Egr2 and Leptin. Additional bioinformatic analyses identified 6 candidate gene markers of CO2 sensitive 5-HT neurons, and 2 selected candidate genes (CD46 and Iba57) were both expressed in 5-HT neurons determined via in situ mRNA hybridization. Together, these data provide novel insights into the transcriptional control of cellular chemoreception and provide unbiased candidate gene markers of CO2 sensitive 5-HT neurons. Methodologically, these data highlight the utility of the patch-to-seq technique in enabling the linkage of gene expression to specific functions, like CO2 chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.

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.