Corn oil vehicle

Male, 8-week-old, C57BL/6J littermate mice were purchased from Damul Science (Daejeon, Korea), housed in a temperature-controlled facility (22 ± 1 °C) that maintained a 12 h light/dark cycle, and provided free access to a standard chow diet (5L79, LabDiet, St. Louis, MO, USA) and water. A schematic representation of the study design is shown in Figure 1A. Mice were randomly assigned to each study group. To prepare the acute heart failure rodent model, mice were administered a single dose of 15 mg/kg doxorubicin (Sigma-Aldrich, Oakville, ON, Canada) or 0.9% sterile saline. To prepare the chronic heart failure model, serial intraperitoneal injections of doxorubicin (2.5 mg/kg) or 0.9% sterile saline were administered every 4 days for 24 days (cumulative dosage, 15 mg/kg). One day after doxorubicin injection, the control mice were gavaged daily with vehicle (corn oil) (Sigma-Aldrich), while the experimental mice were gavaged daily with ARNI (ENTRESTO^®; Novartis, Basel, Switzerland) (68 mg/kg/day), SGLT2 inhibitor (Forxiga^®; AstraZeneca, Cambridge, UK) (1 mg/kg/day), Low-ARNI/SGLT2i (Low-ARNI, 34 mg/kg/day; SGLT2i, 1 mg/kg/day), and ARNI/SGLT2i for 6 weeks. Dosage of ARNI and SGLT2i for our study was based on prior preclinical studies [22 (link),23 (link)]. For the collection of blood and tissue samples, animals were euthanized by isoflurane overdose followed by exsanguination.

The experimental mice were exposed to BPA at post-natal day (PND) 3 to resemble the human final gestational stage and aiming at the critical developmental window of T lymphocytes. Briefly, 72 h after birth the pups were sexed by ano-genital distance. The pups of both genders received treatment, though whole litters were assigned to experimental groups to avoid pup reallocation stress. The control group received no neonatal treatment. The vehicle group received a dorsal subcutaneous injection of 20 μl corn oil vehicle (Sigma, St. Louis MO). The BPA group received 250 μg/kg bw of BPA. Given that neonate rodents have minimal glucuronidation activity, which is the major metabolic mechanism for BPA clearance [16 (link),19 (link)], this dose equals to a brief 5-day exposure according to the FDA reference dose of 50 μg/kg bw/day, performed in a single administration to avoid excessive manipulation stress. Though the main route of exposure is commonly oral, a subcutaneous injection was selected instead as no difference between oral and subcutaneous routes are observed in neonate mice in this case [20 (link)]. The pups were weaned at 21 days of age and placed in standard cages, 5 mice per cage.