Maintaining temporal coordination of physiological function is essential for good health. To determine how the endothelin axis regulates the timing of blood pressure (BP) and key physiological rhythms, we monitored circadian rhythmicity of BP and other variables by telemetry in male and female Sprague-Dawley rats with or without chronic ET_B receptor blockade (A-192621). Rats were maintained under constant dark conditions to ensure a free running status of the circadian clock. Rats were divided into groups being fed either normal salt (NS) or high salt (HS) diets starting the day of release into constant darkness. After one week, a subset of rats was given A-192621 in the food for two weeks. HS diet exacerbated amplitude of mean arterial pressure (MAP) in ET_B antagonist treated rats. ET_B antagonism was sufficient to delay MAP rhythms either diet. There were no changes in amplitude of heart rate (HR) or body temperature resulting in desynchrony between the phases with MAP. Rats fed a HS diet without antagonist also displayed desynchrony among all peripheral rhythms. Together these data support the notion of the endothelial system regulating BP rhythms and maintaining synchrony with peripheral physiological rhythms.

2. Materials and Methods

2.1. Animals and Surgical Preparation

All procedures were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Sprague-Dawley (SD) rats were bred from an in-house colony originally obtained from Charles River Laboratories or purchased from Charles River Laboratories. Purchased SD rats were allowed to acclimate in the in-house colony for one week minimum prior to any interventions. Male (8-10 weeks, 375 ± 16 g body weight) and female SD (10-12 weeks, 266 ± 8 g body weight) rats were implanted with telemetry transmitters (HD-S10, Data Sciences International, Minneapolis, MN) in the abdominal aorta as previously described (Becker et al., 2017a). Animals were allowed to recover 7-10 days post-surgery in a 12-hour light 12-hour dark (12L:12D) cycle. Following recovery, the animals were then released into constant darkness (DD) and fed either a normal salt (NS, 0.49 % NaCl, TD.96208: Envigo, Indianapolis, IN) or high salt diet (HS, 4 % NaCl, TD.92304) for one week to ensure a free running state of the circadian clock. Baseline recordings were taken during the last 72 hours of the first week in DD. After baseline recordings concluded, the animals were further divided into 4 groups: NS vehicle, HS vehicle, NS + ET_B antagonist, A-192621 (PepTech Corp., Bedford, MA), or HS+A-192621. Rats in the vehicle group were given either NS or HS ground diet. The ET_B antagonist-treated rats were given powdered NS or HS with A-192621 mixed in the diet at a concentration to deliver a dose of 10mg/kg/day for 2 weeks (Becker et al. 2021; Pollock and Pollock 2001). Rats were fed ad libitum to avoid any meal timing cues. Animals were then transferred to metabolic cages within the constant dark room to facilitate collections every 12 hours for 2 days. Rats were maintained in the dark room while being sacrificed to allow blood collection. Blood was centrifuged for collection of plasma and stored at -80 °C.

2.2. Telemetry analysis

Telemetry recordings were taken in 1-minute intervals every 10 minutes for 24 hours per day throughout the experiment using Ponemah™ v6.0 (Data Sciences International). Heart rate frequency domain analysis was completed using 30-second bins of low frequency (LF; 0.2-0.6 Hz) and high frequency (HF; 1.0-3.0 Hz) using the maximum slope derivative of the blood pressure signal for the last 48 hours of recordings on the Ponemah software. Data were analyzed offline using pivot tables on Microsoft Excel to obtain 1-minute bins and 1-hour bins. Heart rate frequency domain data were binned into 1-hour segments and averaged across the 2 consecutive days. Cosinor analysis was done on each animal using a custom nonlinear regression equation in GraphPad Prism™ v10.6 (GraphPad Software, LLC) as described in Becker and colleagues to obtain the MESOR, amplitude, and R² (goodness of fit; Becker et al. 2017). Cosinor analysis was conducted on 1-hour bins from the last 6 days of recording of mean arterial pressure (MAP), systolic and diastolic blood pressure (SBP, DBP), body temperature, heart rate (HR), and locomotor activity. 1-minute bins from the last 11 days of MAP, SBP, DBP, HR, body temperature, and locomotor activity recordings were used to determine the free running period (FRP) using the chi-squared periodogram on Clocklab™ (Actimetrics Inc.). Arrhythmic animals with a non-detectible FRP were excluded from FRP and acrophase comparisons. The acrophase was determined for each animal on Cosinor.Online using the FRP for each animal and 1-hour bins from the last 6 days of recordings.

2.3. Metabolic Cage Collections

Animals were placed in metabolic cages and acclimated for 48 hours. Following acclimation, food and water intake measurements were taken every 12 hours for 48 hours. Urine was collected at these time intervals for analysis. Subjective day (inactive period) and subjective night (active period) were determined by using a minimum of 17 days of body temperature telemetry recordings in constant darkness and determining the acrophase of the day in Clocklab. A linear regression line was drawn to predict the acrophase for the coming day. The acrophase of body temperature in rats peak during the subjective night, therefore collections done at the end of the subjective night/ beginning of subjective day were classified as measurements taken during circadian time (CT) CT0-CT3. CT0-CT3 was calculated by the equation acrophase + (FRP/4). Oppositely, CT12-CT15 was classified as the end of the subjective night period and calculated by the acrophase + (FRP/4).

2.4. Urinary Analysis

Urine was collected and weighed to determine total urinary volume excreted. Urinary collections were then separated into 1 ml aliquots and stored in -20 °C until analysis. Urinary Na⁺ and K⁺ concentrations were determined by ion-selective electrodes (Easylyte™, Medical Corporation). Urea concentrations were measured via colorimetric assay (QuantiChrom™, Abcam). Urinary excretion rates were determined by multiplying urine concentration by the 12-hour urinary volumes.

2.5. Statistical analysis

Circadian data used for acrophase measurements were analyzed via cosinor analysis on Cosinor.Online and done individually using the FRP from each animal. Otherwise, data were analyzed using a cosinor analysis on Graph Pad Prism (Becker et al. 2017). All parameters were tested for normality. If normally distributed, groups were compared using a two-way ANOVA followed by a Tukey’s post-hoc. If data were not normal, an aligned rank ANOVA (ART ANOVA) was performed. Rayleigh plots were made using Oriana™ software (Kovach Computing Services, UK) and differences were tested for using a Watson’s F test. If the p value was < 0.05 then Watson U² tests were used for pairwise comparisons.