Adult Rat Cardiomyocyte Isolation and Culturing

Cardiomyocytes were isolated from adult Wistar rats (n = 5) weighing 200–250 g. Intact AT (left and right atrium were combined), LV, RV, and IVS rat cardiomyocytes were isolated through collagenase digestion of hearts essentially as described previously (Sequeira et al., 2015; van Deel et al., 2017). In brief, rats were euthanized via isoflurane inhalation, after which hearts were quickly harvested and washed in cold isolation Tyrode solution (130 mM NaCl, 5.4 mM KCl, 3 mM sodium pyruvate, 25 mM HEPES, 0.5 mM MgCl₂, 0.33 mM NaH₂PO₄, 22 mM glucose, pH 7.4) containing 0.2 mM EGTA (Tyrode–EGTA). Hearts were subsequently cannulated to a Langendorff set-up via the aorta and perfused with Tyrode-EGTA for 2 min at 37°C. Next, hearts were perfused for 25–35 min (depending on the integrity of the tissue) with enzyme Tyrode solution composed of Tyrode solution, 50 μM CaCl₂, and 1.2 mg/mL collagenase (Type II, 265 U/mg; Worthington Biochemical, NJ, United States). The heart was removed from the cannula and separated into AT, LV, RV, and IVS. Each part was cut into fine pieces, followed by trituration for 3 min with a plastic Pasteur pipette in stopping buffer solution 1 [SB-1; Tyrode solution, 0.6 mg/mL collagenase, 100 μM CaCl₂, and 10 mg/mL bovine serum albumin (BSA)]. Cell suspensions were filtered through a 300 μm nylon mesh filter and collected in 50 mL Falcon tubes, followed by centrifugation for 1 min at 27 × g at room temperature. Pellets containing cardiomyocytes were resuspended in SB solution 2 (SB-2; Tyrode solution, 250 μM CaCl₂ and 10 mg/mL BSA) and incubated for 10 min at 37°C in order for the cells to settle. After the removal of supernatants, cardiomyocytes were resuspended in SB solution 3 (SB-3; Tyrode solution, 500 μM CaCl₂ and 10 mg/mL BSA) and incubated for 10 min at 37°C. Hereafter, cardiomyocytes were suspended in plating medium [Medium 199 (Lonza, Basel, Switzerland), 1% penicillin/streptomycin and 5% fetal bovine serum (FBS)] and transferred to laminin-coated 35 mm² glass-bottomed dishes (MatTek, Ashland, MA, United States). Following 1 h of incubation at 37°C in humidified air with 5% CO₂, unattached cells were washed away by replacing plating medium with culture medium [Medium 199, 1% penicillin/streptomycin, insulin transferrin selenium (ITS; Sigma-Aldrich, composition: insulin, 10 mg/L; transferrin, 5.5 mg/L; selenium 5 μg/L) and 0.5 μM cytochalasin D (Life Technologies)]. The latter two compounds were added in order to prevent dedifferentiation and optimize cell viability (Viero et al., 2008; Tian et al., 2012). Cardiomyocytes were cultured overnight in an incubator at 37°C in humidified air with 5% CO₂.