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 1 A. 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.
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