Mice (7 months old) were trained to be grasped because transthoracic echocardiography was performed in nonsedated mice to avoid any cardiac depressor effect of anesthetic agents. Typical heart rates at recording were above 600 beats per minute (bpm). Mice were carefully caught by the left hand and placed in supine position. Images were acquired from a parasternal position at the level of the papillary muscles using a 13-MHz linear-array transducer with a digital ultrasound system (Vivid 7, GE Medical System, Horton, Norway). Left ventricular (LV) diameters and anterior and posterior wall thicknesses were serially obtained from M-mode acquisition. As we expected a homogenous function and planned to compare sequential echocardiography, we used M-mode technique to assess both LV volumes, mass, and ejection fraction. Relative LV wall thickness was defined as the sum of septal and posterior wall thickness over LV end-diastolic diameter, and LV mass was determined using the uncorrected cube assumption formula [LV mass = (AWTdþ LVEDDþ PWTd)3 − (LVEDD)3]. Diastolic function was not assessed by echocardiography because of the heart rate above 600 bpm precluding the analysis of transmitral flow. Therefore, we assessed relaxation by dP/dtmin during in vivo hemodynamic analysis. Peak systolic values of radial Strain rate (SR) in the anterior and posterior wall were obtained using tissue Doppler imaging (TDI) as previously described (47). TDI loops were acquired from the same parasternal view with a careful alignment with the radial component of the deformation (48) at a mean frame rate of 514 frames per second and a depth of 1 cm. The Nyquist velocity limit was set at 12 cm/s. Radial SR analysis was performed offline using the EchoPac Software (GE Medical Systems) by a single observer (G.D.) blinded to the genotype of the animals. Peak systolic of radial SR was computed from a region of interest positioned in the mid-anterior wall and was measured over an axial distance of 0.6 mm. The temporal smoothing filters were turned off for all measurements. Because slight respiratory variations exist, we averaged peak systolic of radial SR on eight consecutive cardiac cycles. The intra-observer variability of radial SR was assessed (G.D.) using the same acquisition and same method at 24-hour intervals [3.5 ± 3.4% (3.3 to 3.7)].

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