Langendorff-perfused rat hearts

Rat hearts were Langendorff-perfused as described previously⁶⁶. Briefly, Wistar rats (Rattus norvegicus, male, 200–250 g, 7 weeks old) were anesthetized by injection of 0.9 mL sodium pentobarbitone 20% (w/v). Hearts were removed immediately and washed free of blood in ice-cold Krebs-Henseleit solution (composition in mmol L⁻¹: 114 NaCl, 5.9 KCl, 1.16 MgSO₄, 25 NaHCO₃, 0.48 EDTA, 2.2 CaCl₂, 5 glucose, 1 Na L-lactate, 0.1 Na-pyruvate, 0.5 L-glutamic acid, 4 hydroxybutyrate, pH 7.4) supplemented with 10 μmol L⁻¹ insulin and 0.04% (v/v) intralipid. The aorta was cannulated and secured with sutures (Mersilk 3‐0; Ethicon, Somerville, NJ, USA). Hearts were perfused at a constant perfusion pressure of 70 ± 2 mmHg using a peristaltic pump (Gilson Minipuls 4, Middleton, WI, USA) and a feedback control system (STH Pump Controller; AD Instruments, Oxford, UK). Buffer was equilibrated with 95% O2–5% CO2 using a custom‐made counter‐current membrane oxygenator consisting of spirally wound Silastic tubing (1.47 mm i.d., 1.96 mm o.d.; VWR International, Lutterworth, UK) continually flushed with gas at 37 °C. A fluid‐filled balloon, attached to a pressure transducer, was inserted into the left ventricle and inflated to give an end‐diastolic pressure between 3 and 8 mmHg. Left ventricular pressure (LVP), perfusion pressure, coronary flow and arterial and venous oxygen tensions were recorded using a PowerLab recorder and LabChart 8.0 software (ADInstruments). Functional parameters were averaged for ∼80 cardiac cycles at 5 min intervals.

All animals were treated in accordance with the guidelines approved by the UK Animal Scientific procedures Act (1986) and European Union Directive 2010/63/EU. All procedures were performed according to Schedule 1 of the UK Animal Scientific Procedure Act, 1986, which do not require ethical approval. All procedures complied with the relevant ethical regulations and were carried out in accordance with the guidelines of the Animal Welfare and Ethical Review Body (AWERB, King’s College London).