4.1. Rat Heart Failure Model

All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The experiments were designed according to the guidelines from the Federation of European Laboratory Animal Science Associations (FELASA), EU animal research directive (86/609/EEC) and Council of Europe (ETS 123) and the EU directive (2010/63/EU) and approved by the national animal ethics commitee, 4283. The 3 R’s (replacement, reduction, and refinement) have specifically been addressed when designing the study.

Rats were anesthetized with 5% isoflurane, then intubated and ventilated with 1.5% isoflurane in a 30% O₂/70% N₂O mixture. HF was induced by ligation of the descending coronary artery, leading to large myocardial infarction (MI) in the left ventricle (LV), as previously described [52,53]. After left thoracotomy, the pericardium was opened, and in the MI group, the descending artery was ligated with a polyester suture (Ethibond 6-0, needle Rb-2, Ethicon; Norderstedt, Germany). Sham rats underwent the same surgical procedure, except for ligation of the descending coronary artery. Buprenorfin (0.04 mg/kg) was injected subcutaneously during the surgery and repeated 8 h thereafter to relieve pain. After four weeks, rats with MI operation were examined by echocardiography to determine the extent of MI. Only rats with an MI size of 40–50% of the left ventricle were included, which gave an ejection fraction of about 20% (data not shown).