2.2. Transthoracic Echocardiography

Transthoracic echocardiography was conducted using the SonoScape S2V high-resolution color Doppler ultrasound system equipped with a 9 MHz C611 transducer (SonoScape Co., Shenzhen, China) which is specifically designed for mice and rats. Just before sacrifice, mice were anesthetized with isoflurane where 3% was used for induction and 1.5% for maintenance, at a flow of 1 L/min using an EZ-SA800 Anesthesia Single Animal System (E-Z Systems, Pennsylvania, USA). Left ventricular (LV) parasternal long-axis 2D view in M-mode was performed at the level of papillary muscle to assess LV wall thicknesses and internal diameters, allowing the calculation of the fractional shortening (FS) and ejection fraction (EF) by the Teicholz method. EF (%) was calculated using the following formula: EF = (EDV − ESV)/EDV ×100; EDV: end-diastolic volume and ESV: end-systolic volume. FS (%) was calculated based on the diameters of the left ventricle: FS = (LVIDd − LVIDs)/LVIDd × 100; LVIDd: end diastolic left ventricular internal diameter and LVIDs: end systolic left ventricular internal diameter. Experiments were done by two independent operators blinded to the conditions.