Animals

Wild-type (WT, C57BL/6J, DBA/2, 129Sv) mice were used for this study after regeneration from sperm cryopreserved in straws with C57Bl/6J females. Mice were housed in central animal facility of the Faculty of Medicine in Nancy. The French Animal Care Committee, in accordance with European regulations, approved all protocols (n°APAFIS#34184). Details on the origin of WT and knockout β1-AR−/− mice are described in the supplementary methods.

Septic shock modelCLP surgery

Septic shock was induced by cecal ligation and puncture (CLP) in both male and female mice aged 12–16 weeks, weighing 20–40 g. In non-treated groups (SHAM), mice only underwent a laparotomy with exposure of the caecum. The detailed procedure, performed according to the literature, is presented in Supplemental Data Material—Methods [19].

Resuscitation

Three hours after the surgery, the mice received an intraperitoneal fluid bolus (5 ml/100 g, 0.9% NaCl) containing a broad-spectrum antibiotic therapy (0.01 mg/g, imipenem 500 mg, cilastatine 500 mg, Tienam, MSD, Kenilworth). All mice received the same amount of fluid per unit of body weight.

β3-AR agonist and antagonist treatments

β3-AR agonist (CL316243, a phenylethanolamine: C5976, Merck KGaA, Darmstadt, Germany) and β3-AR antagonist (SR59230A, an aryloxypropanolaminotetraline: S8688, Merck KGaA, Darmstadt, Germany) were administered once at 0.001 mg/g jointly with the fluid bolus three hours after the surgery. The doses of agonists and antagonists were selected based on literature from studies using a murine model of sepsis [20].

Experimental design

The mice were acclimatized for 5 days in the experimental area. Following surgery, they were randomly assigned to one of four groups: SHAM, CLP, CLP+ agonist, and CLP+ antagonist. Three hours after waking up from surgery, mice were resuscitated and received the interventions (agonist or antagonist). Then, mice were placed in a specific post-operative area, at room temperature, and they were left to rest for 15 h. Eighteen hours post‐CLP, mice underwent measurements and experimentations. Of note, the number of mice used and reasons for failed measurements and for exclusion are provided in Supplemental Data Material—Table S1.

Measurements and experimental protocolsEchocardiography

Transthoracic echocardiography was performed using the VEVO3100 (FUJIFILM VisualSonics, Toronto, Canada). A full description is provided in Supplemental Data Material—Methods. Mice were placed on a heating pad and the analysis began when mice temperature reached 36 °C. Mean arterial pressure (MAP) was measured through a catheter inserted into the left carotid artery with a blood pressure transducer (ACQ7700, DSI Harvard Biosciences, Holliston, USA).

Vascular reactivity procedure

Mice were killed, the thoracic aorta and mesenteric artery were collected and their vasoreactivity was studied by ex vivo myography. The procedure is detailed in Supplemental Data Material—Methods.

Immunostaining

The aortic tissue was embedded in O.C.T. (Lamb/OCT, ThermoFisher Scientific, Waltham, USA) and flash frozen in liquid nitrogen. Six-micrometer cryosections were performed using a cryostat (Leica Microsystems, Wetzlar, Germany). After fixation with 4% paraformaldehyde, the histological sections of aorta were labeled with the primary antibodies (Supplemental Data Material—Table S2) of interest, and then detected with fluorescent secondary antibodies.

Quantifications were carried out using ImageJ software (National Institutes of Health, USA). The images were not analyzed in a blinded manner. The area of interest was manually traced and measured in square micrometers. The integrated density was determined for each traced area. Background fluorescence intensity was measured. Corrected Tissue Fluorescence (CTF) was calculated using the following formula:

$$ } = }\;}\left( }\;}\;}\;} \times }\;}\;}\;}\;}} \right). $$

The calculation of the proportions from the CTF values was carried out in this way per animal for each group:

$$ \frac}\;}\;}\;}\;}\;}\upbeta }}}}\upbeta }}} \times 100 $$

Polymerase chain reaction

Total RNA extraction was carried out with the RNA Plus mini-Kit (74104, Qiagen NV, Venlo, The Netherlands) according to the manufacturer’s instructions. The extracted RNAs were reverse transcribed into cDNA using the kit manufacturer’s instructions and in BioRad iCycler iQTM (1708891, BioRad, Hercules, USA). The sequences of interest, described in Supplemental Data Material—Table S3), were hybridized to the probes and amplified with the BioRad CFX Connect-Real-Time System (Hercules, USA).

Western blot

Proteins from the heart, thoracic aorta and mesenteric arteries were extracted using a lysis buffer containing 25 mM bicine buffer (pH 7.6) with phosphatase inhibitor and complete protease inhibitor reagents (04719956001, Roche, Basel, Switzerland). The protein concentration was determined using the Bradford reagent. After separation on SDS-PAGE gels, proteins were transferred to nitrocellulose membranes, which were incubated overnight at 4 °C with primary antibodies (Supplemental Data Material–Table S4) after a blocking step. A chemiluminescent signal was produced using ECL+ solution (170-5060, BioRad Hercules, USA) and detected using a Fusion FX imager (Vilber, Marne-la-Vallée, France). Relative densitometry was performed using Image MultiGauge 3.0 software (Fujifilm, Tokyo, Japan). Protein normalization was conducted using the total protein method with Ponceau Red staining.

Survival study

Survival was studied in CLP, CLP+ agonist and antagonist groups. The animal census was conducted twice a day for 5 days. Administration of agonist or antagonist began three hours post-CLP and was reinjected intraperitoneally daily (for both agents, half-life = 16 h and dose = 0.001 mg/g) with buprenorphine (dose = 0.0002 mg/g) in a bolus of 200 µL (0.7 ml/100 g). The clinical severity score and weight of each mouse were measured daily. This score includes several items such as appearance, provoked behavior, unprovoked behavior and hydration status (Supplemental Data Material—Table S5). This clinical score ranging from 0 to 15 was employed to evaluate the daily condition of the mice. The endpoint to decide euthanasia for the 5-day survival study was defined as a clinical score > 10 (Supplemental data material—Table S5), in combined with a weight loss greater than 10%.

Sample size

The sample size was determined, in accordance with the Animal Ethics Committee, on our previous study employing the same murine CLP model [7]. Consequently, eight animals per group were deemed necessary.

Statistics

Data in the tables are presented as medians with 25th and 75th percentiles, or with min–max when n < 5. Comparisons between the SHAM and CLP groups were presented using Hodges–Lehmann estimates with 95% confidence intervals and no further tests were performed for vasoreactivity, western blot and RT-PCR analyses. Comparisons among the CLP, CLP+ agonist, and CLP+ antagonist groups in tables and figures were performed using the Kruskal–Wallis test. Post-hoc pairwise comparisons were conducted using Dunn’s test. Kaplan–Meier curves were plotted for the survival study, and the log-rank test was used for comparisons. Analyses of weight changes and clinical severity scores were performed using a linear mixed model with random effects for each mouse and time point. The assumption of normality for the residuals was assessed using both the quantile–quantile (Q–Q) plot and the W statistic from the Shapiro–Wilk test. No statistical tests were performed when n < 5. A p value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, CA) and R version 4.3.3 (2024-02-29).