In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared to normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in Drp1 and a decrease in Mfn2 expression, mitochondrial fission and fusion proteins, respectively. Pro-inflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of pro-inflammatory cytokines in mitochondrial remodeling is not clearly established. In non-asthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in 3-D using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor, aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased PGC1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O2 consumption rate. However, when normalized for mitochondrial volume density, O2 consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O2 consumption rate per mitochondrion.

In aged rats, diaphragm muscle (DIAm) reduced specific force and fiber cross-sectional area, sarcopenia, is selective for vulnerable type IIx and/or IIb DIAm fibers, with type I and IIa fibers being resilient. In humans, the oxidative capacity [as measured by maximum succinate dehydrogenase (SDHmax) activity] of fast-type muscle is reduced with aging, with slow-type muscle being unaffected. We hypothesized that in aged Fischer rat DIAm exhibiting sarcopenia, reduced SDHmax activity would occur in type IIx and/or IIb fibers. Rats obtained from the NIA colony (6, 18, and 24 mo old) were euthanized, and ~2-mm-wide DIAm strips were obtained. For SDHmax and fiber type assessments, DIAm strips were stretched (approximately optimal length), fresh frozen in isopentane, and sectioned on a cryostat at 6 μm. SDHmax, quantified by intensity of nitroblue tetrazolium diformazan precipitation, was assessed in a fiber type-specific manner by comparing serial sections labeled with myosin heavy chain (MyHC) antibodies differentiating type I (MyHCSlow), IIa (MyHC2A), and IIx and/or IIb fibers. Isometric DIAm force and fatigue were assessed in DIAm strips by muscle stimulation with supramaximal pulses at a variety of frequencies (5–100 Hz) delivered in 1-s trains. By 24 mo, DIAm sarcopenia was apparent and SDHmax in type IIx and/or IIb fibers activity was reduced ~35% compared with 6-mo-old control DIAm. These results underscore the remarkable fiber type selectivity of type IIx and/or IIb fibers to age-associated perturbations and suggest that reduced mitochondrial oxidative capacity is associated with DIAm sarcopenia. NEW & NOTEWORTHY We examined the oxidative capacity as measured by maximum succinate dehydrogenase activity in older (18 or 24 mo old) Fischer 344 rat diaphragm muscle (DIAm) compared with young rats (6 mo old). In 24-mo-old rats, SDH activity was reduced in type IIx/b DIAm fibers. These SDH changes were concomitant with sarcopenia (reduced specific force and atrophy of type IIx/b DIAm fibers) at 24 mo old. At 18 mo old, there was no change in SDH activity and no evidence of sarcopenia.

Activation of central AT 1 Rs (angiotensin type 1 receptors) is required for the increased blood pressure, polydipsia, and salt intake in deoxycorticosterone acetate (DOCA)–salt hypertension. TRV120027 (TRV027) is an AT 1 R-biased agonist that selectively acts through β-arrestin. We hypothesized that intracerebroventricular administration of TRV027 would ameliorate the effects of DOCA-salt. In a neuronal cell line, TRV027 induced AT 1a R internalization through dynamin and clathrin-mediated endocytosis. We next evaluated the effect of chronic intracerebroventricular infusion of TRV027 on fluid intake. We measured the relative intake of water versus various saline solutions using a 2-bottle choice paradigm in mice subjected to DOCA with a concomitant intracerebroventricular infusion of either vehicle, TRV027, or losartan. Sham mice received intracerebroventricular vehicle without DOCA. TRV027 potentiated DOCA-induced water intake in the presence or absence of saline. TRV027 and losartan both increased the aversion for saline—an effect particularly pronounced for highly aversive saline solutions. Intracerebroventricular Ang (angiotensin) II, but not TRV027, increased water and saline intake in the absence of DOCA. In a separate cohort, blood pressure responses to acute intracerebroventricular injection of vehicle, TRV, or losartan were measured by radiotelemetry in mice with established DOCA-salt hypertension. Central administration of intracerebroventricular TRV027 or losartan each caused a significant and similar reduction of blood pressure and heart rate. We conclude that administration of TRV027 a selective β-arrestin biased agonist directly into the brain increases aversion to saline and lowers blood pressure in a model of salt-sensitive hypertension. These data suggest that selective activation of AT 1 R β-arrestin pathways may be exploitable therapeutically.

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 β-arrestin mediated signaling, but the contributions of each remain largely unexplored. Here, we hypothesized that β-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 β-arrestin1 ( Arrb1 ) and β-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 (β-arrestin 1), might counterbalance the canonical signaling of GPCRs.

The renin-angiotensin system (RAS) within the brain is implicated in 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 upon 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. Further, 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.

The brain renin angiotensin system (RAS) is known for its role in cardiovascular and metabolic regulation. Angiotensin II (Ang II) is the major active product of the RAS, exerting most of its physiological effects through the angiotensin type-1 receptor (AT 1 R). Canonical or G-protein-mediated signaling of the AT 1 R within the brain has long been known to induce a dipsogenic and pressor response upon Ang II stimulation. Non-canonical or β-Arrestin mediated signaling is thought to counterbalance the detrimental effects of canonical signaling. However, the non-canonical AT 1 R/β-Arrestin pathway within the brain is understudied. Therefore, it is hypothesized that β-Arrestin activation contributes to fluid homeostasis and blood pressure (BP) regulation. Global β-Arrestin1 ( Arrb 1) and β-Arrestin2 ( Arrb 2) knockout (KO) mice were employed to evaluate drinking behavior and BP with and without deoxycorticosterone acetate (DOCA). Age- and sex-matched C57BL/6J mice served as controls. Mice were subjected to the two-bottle choice paradigm, in which the animals were presented with two bottles, one containing water and one containing 0.15M saline. In the absence of DOCA, mice lacking β-Arrestin2 had increased saline intake when compared to β-Arrestin1-KO and wildtype (WT=2.2±0.2 and Arrb 1-KO=2±0.4 vs Arrb 2-KO=5±0.7 mL/day; p<0.001; n=13, 11 and 9, respectively). This resulted in a saline preference, which means mice preferred saline over water by more than 50% by volume. In the presence of DOCA, mice lacking β-Arrestin2 had increased saline intake when compared to β-Arrestin1-KO and wildtype (WT=10.6±1.2 and Arrb 1-KO=6.5±0.8 vs Arrb 2-KO=16.6±2 mL/day; p<0.001; n=13, 11 and 9, respectively). However, these mice did not develop a saline preference. Preliminarily, β-Arrestin2-KO mice exhibited higher BP when compared to WT at baseline (WT=108±5 vs Arrb 2-KO=124±6 mmHg; n=2), which was exacerbated in response to DOCA (WT=122±6 vs Arrb 2-KO=140±5 mmHg; n=2). These findings suggest that β-Arrestin2 might counterbalance effects of canonical activation of the AT 1 R through G proteins. Overall, β-Arrestin2 appears to protect against cardiovascular diseases since the genetic ablation of β-Arrestin2 resulted in an increase in saline intake and exacerbated BP.

No abstract

Low-renin hypertension is a common subset of hypertensive patients, which is associated with salt sensitivity. Deoxycorticosterone (DOCA)-salt hypertension, a prototypical model of low-renin hypertension, requires activation of the angiotensin type-1 receptor (AT1R) in specific brain areas. G protein-mediated signaling of the AT1R within the brain is known to induce dipsogenic and pressor responses to Ang II stimulation. Non-canonical or b-arrestin-mediated signaling is thought to counterbalance the maladaptive G-protein (Gαq) signaling during disease. Recently, we found that global deletion of β-arrestin2 (ARRB2) exhibited an exacerbated increase in blood pressure (BP) in response to DOCA-salt compared to WT suggesting a protective role for ARRB2. However, the role of the non-canonical AT1R/β-arrestin pathway within the brain is understudied. Consequently, we hypothesized that b-Arrestin activation within the brain contributes to BP regulation. Global male and female β-arrestin1 ( Arrb1)- and β-arrestin2 ( Arrb2)-KO mice were employed to evaluate acute BP response upon treatment with intracerebroventricular (ICV) infusion of AngII (1ug). Age- and sex-matched C57BL/6 mice served as controls. Mice were simultaneously instrumented with radiotelemeters and ICV-cannula, BP was continuously recorded before and after AngII infusion in awake animals. At baseline, Arrb2-KO mice showed a slight increase in BP WT (WT=134.0±10.7 vs Arrb2-KO=143.7±17.2 mmHg; n=14 and 19, respectively). Further, Arrb2-KO mice exhibited significantly higher BP in response to AngII (WT=143.7±17.2 vs Arrb2-KO=176.9±24.2 mmHg; n=14 and 19, respectively p<0.05). These findings suggest that loss of ARRB2 exacerbates the pressor response to AngII within the brain. Additionally, we evaluated BP upon activation β-arrestin using TRV120027 (TRV27), a β-arrestin biased agonist specific for the AT1R. We used the BPH2/J genetic mouse model of low renin hypertension. Mice were subjected to continuous infusion of TRV27 (6ug/h), and telemetric BP was continuously recorded. BPN/2J mice were used as the normotensive controls. At baseline, BPH2/J exhibited significantly higher BP when compared to normotensive controls (BPN=123.9±5.3 vs. BPH=153.4±3.4 mmHg; n=4 p<0.05). Further, chronic delivery of ICV-TRV showed a higher magnitude of BP reduction in the BPH2/J group compared to controls (BPN=-3.8±3.0 vs. BPH=-8.8±1.3 mmHg; n=4 p<0.05), suggesting that the effects of b-arrestin activation are predominant under hypertensive states. Overall, endogenous ARRB2 counterbalances the deleterious effects of AT1R overactivation in the brain. The stimulation of the AT1R β-arrestin axis in the brain using biased AT1R agonist that penetrate the brain-blood barrier might be a potential strategy to treat low renin hypertension. HL084207 and HL144807 to CDS, and 22PRE898004 to NMM This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.