Male 8- to 12-week-old mice weighing 22–28 g underwent permanent left anterior descending artery ligation or sham operation as previously described (52 (link)). Briefly, mice were anesthetized with 4% isoflurane and intubated with a 20-gauge cannula. Ventilation was initiated and continued under 2 % isoflurane at 130 breaths per minute by a small-animal TOPO ventilator (Kent Scientific). A small skin cut (~1.2 cm) was made over the left chest. After dissection and retraction of the pectoral major and minor muscle, the fourth intercostal space was exposed. A small hole was made at the fourth intercostal space, and a mosquito clamp was used to open the pleural membrane and pericardium. With the clamp slightly open, the heart was smoothly and gently popped out through the hole. The left coronary artery was located, sutured, and ligated at a site about 3 mm from its origin using a 7-0 Ethilon silk suture. After ligation, the heart was immediately placed back into the intrathoracic space, followed by manual evacuation of air and closure of muscle and the skin, by means of the previously placed purse-string suture. Mice that did not survive the first 24 hours after the surgery were excluded from analysis. Sham-operated animals underwent the same procedure without coronary artery ligation.
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