Echocardiography was performed to evaluate the cardiac geometry, and systolic and diastolic function as previously described 23. The Visual Sonics high-resolution Vevo2100 ultrasound system (VisualSonics Inc., Canada) with a 30-MHz linear array ultrasound transducer (MS-400, VisualSonics Inc.) was used. In brief, mice were anesthetized with isoflurane (2% vol/vol) with a rodent respirator device until the heart rate stabilized at 400 to 500 beats per min. Parasternal long-axis images were acquired in B-mode with appropriate position of the scan head to identify the maximum LV length. In this view, the M-mode cursor was positioned perpendicular to the maximum LV dimension in end-diastole and systole, and M-mode images were obtained for measuring wall thickness and chamber dimensions. LV ejection fraction (LVEF) and LV fractional shortening (LVFS) were calculated automatically. The apical four-chamber view was acquired and the peak flow velocities during early diastole (E wave) and late diastole (A wave) across the mitral valve, as well as early-diastolic peak velocity (e') of the mitral valve ring were measured. E/e' and E/A ratios, which reflected the left ventricular diastolic function, were calculated.

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