2.2. Neonatal Rat Cardiomyocyte (NRCM) Isolation and Culture

As previously described [22], NRCMs were isolated from the heart of 1- to 3-day-old Wistar rats. Extirpated hearts were minced, and ventricles were digested four times for fifteen minutes each in trypsin (0.25% Invitrogen, Carlsbad, CA, USA) in a sterile environment. Cells were incubated for 90 minutes in cell culture flasks to allow noncardiac myocytes (mainly cardiac fibroblasts) to adhere to the plastic. NRCMs were cultured in F-10 medium (1x) nutrient mixture (HAM) with L-glutamine (Gibco, Waltham, MA, USA) containing 5.5 mmol/L of D-glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 100 U/mL of penicillin, and 100 mg/L of streptomycin (Gibco, Waltham, MA, USA). NRCMs (1 × 10⁶ cells) were placed in a six-well culture plate and incubated at 37°C in a humidified atmosphere (5% CO₂/95% O₂). Experiments were performed on beating and confluent monolayers on the 3rd to 5th day of culture. Cell cultures were subdivided into the following experimental groups: (1) control treated with vehicle DMSO (0.1%) (control-DMSO), (2) control treated with fenofibrate (10 μM) (control-Feno), (3) control treated with mannitol (19.5 mM) (control-mannitol), (4) hypoxia/reperfusion treated with DMSO (0.1%) (H/R-DMSO), (5) H/R treated with fenofibrate (10 μM) (H/R-Feno), (6) high glucose (25 mM) treated with DMSO (0.1%) (HG-DMSO), (7) high glucose (25 mM) treated with fenofibrate (10 μM) (HG-Feno), (8) high glucose (25 mM) plus hypoxia/reperfusion treated with DMSO (0.1%) (HG+H/R-DMSO), and (9) high glucose 25 mM plus hypoxia/reperfusion treated with fenofibrate (10 μM) (HG+H/R-Feno) (Figure 1). High glucose was produced by incubating cells for 48 hours in F-10 medium containing 25 mmol/L of glucose ((+)D-glucose at 200 g/L, Gibco, Waltham, MA, USA). To produce H/R, cell cultures were covered with a coverslip for two hours [23, 24]. After that, the coverslip was removed and the cells were reoxygenated for 1 hour. Vehicle (DMSO, 0.1%) or fenofibrate (10 μM, purity ≥ 99%, Sigma-Aldrich, St. Louis, MO, USA) were administered according to Figure 1.

Schematic representation of the experimental group design. Primary cardiomyocyte cultures were divided into 9 groups. Mannitol (19.5 mM) and high glucose (25 mM) were administered 48 hrs before fenofibrate (10 μM) or DMSO (0.1%) treatment. Exposition time for either fenofibrate or DMSO treatment was 4 hrs, with or without hypoxia-reperfusion maneuver, before harvest. CT = control; H/R = hypoxia-reperfusion; HG = high glucose; DMSO = dimethylsulfoxide 0.1%; Feno = fenofibrate 10 μM. Yellow bar represents mannitol-treatment period, black bar represents glucose-treatment period, white bar represents hypoxia period, and dashed bar represents reoxygenation period.

In order to explore hyperosmolarity, cells were exposed to mannitol (19.5 mM, D-mannitol, Sigma-Aldrich, St. Louis, MO, USA).