Transverse aortic constriction surgery and echocardiography

Transgenic male mice at ~ 12 weeks of age were injected with tamoxifen to excise the floxed STOP sequence to create cardiac-specific transgenic mice (Fig. ​(Fig.1a).1a). At ~ 16–17 weeks, 6–12 mice of cardiac-specific Raptor het, 4EBP1-Tg, and 4EBP1-mut and WT littermates underwent TAC surgery 3 weeks after tamoxifen induction of the genetic modification. TAC was performed as described (Kim et al. 2008; Tarnavski et al. 2004), under ketamine (130 mg/kg, IP) and xylazine (8.8 mg/kg, IP) anesthesia, supported by a ventilator. Briefly, the skin was incised at the 3th~6th intercostal space, followed by successive layers of subcutaneous tissue/intercostal muscle dissection. A sterile ligature was passed around the exposed aortic arch; then, a blunted 26 gauge needle was placed on top of the aorta; the ligation was tied around the needle, and then, the needle was immediately removed. Echocardiography was performed at baseline and at the end of experiments (4 weeks after TAC) using a Siemens Acuson CV-70 equipped with 13-MHz probe, as described (Dai et al. 2009). Briefly, isoflurane 0.5% mixed with O₂ was used to provide adequate sedation but minimal cardiac suppression during echocardiography. M-mode, conventional and Tissue Doppler echocardiography, and functional calculations were performed according to American Society of Echocardiography guidelines. Myocardial performance index (MPI) is the ratio of isovolemic time (isovolemic contraction and relaxation time) to effective aortic ejection time. Higher MPI indicates worse myocardial performance, as longer time is needed during the isovolemic (ineffective) phase to cope with the change in pressure. Mice were harvested 4 weeks after TAC by cervical dislocation. The heart was removed immediately, rinsed quickly in cold saline, and then flash-frozen in liquid nitrogen.