Experimental perfusion protocol. The hearts were aerobically Langendorff-perfused at a constant pressure equivalent to 80 mmHg during the experiment using 20 min of stabilization before global ischaemia, followed by 60 min of reperfusion after global ischaemia. (A) Preliminary study. The hearts (n = 6/group) were infused with a different dose of drags before 40 min of global ischaemia. Protocol abbreviations: (i) control, no treatment before 40 min of global ischaemia; (ii) DMSO, 0.1% DMSO before 40 min of global ischaemia; (iii) 0.01 µM Esax; (iv) 0.1 µM Esax; and (v) 1.0 µM Esax, administered for 10 min before 40 min of global ischaemia. (B) Study 1. The hearts (n = 6/group) were infused with 0.1 µM esaxerenone before or after 30 min of global ischaemia. Protocol abbreviations: (i) control, no treatment before 30 min of global ischaemia; (ii) pre-Esax, 0.1 µM of Esax administered for 10 min before 30 min of global ischaemia; and (iii) post-Esax, 0.1 µM of Esax administered for 10 min after 30 min of global ischaemia. (C) Study 2. The hearts (n = 6/group) were infused with 1 µM of acetylcholine after 60 min of reperfusion. Protocol abbreviations: (i) Sham, perfusion only; (ii) control, no treatment with 30 min of global ischaemia; and (iii) Esax, 0.1 µM of Esax administered for 10 min before 30 min of global ischaemia. (D) Study 3. The hearts (n = 6/group) were subjected to 30 min of global ischaemia. Protocol abbreviations: (i) control, no treatment; (ii) 5HD, 50 µM of 5HD administered for 10 min before 30 min of global ischaemia; (iii) Esax, 0.1 µM of Esax administered for 10 min before 30 min of global ischaemia; and (iv) Esax-5HD, 0.1 µM of Esax administered with 50 µM of 5HD for 10 min before 30 min of global ischaemia. Esax: esaxerenone; 5HD: 5-hydroxydecanoate.