*4.4. Human Echocardiography*

Echocardiographic examination and analysis were performed by a single operator (BSB) using a GE Vivid Q ultrasound machine equipped with a 3.5 MHz transducer (GE Healthcare, Boston, MA, USA). Images were acquired at rest in the left lateral decubitus position from standard parasternal and apical views. Digital loops of 3 cardiac cycles with associated electrocardiogram information were stored on the hard disk of the ultrasound machine and transferred to a GE EchoPac workstation (GE Healthcare, Boston, MA, USA) for offline analysis. Analysis was performed according to existing guidelines [33]. Interventricular septum thickness, left ventricular posterior wall thickness, and left ventricular systolic and diastolic diameter were measured in the parasternal long axis view. Left atrial volume (LAV) and left ventricular volume in diastole (LVEDV) were calculated from apical views. Left ventricular mass was calculated using the Devereux formula 0.8(1.04[([LVEDD + IVSd + PWd]<sup>3</sup> − LVEDD3)]) + 0.6v, where LVEDD is left ventricular end diastolic diameter, IVSd is thickness of the intraventricular septum in diastole, and PWd is posterior wall thickness in diastole. Relative wall thickness was calculated with the formula (2\*PWd)/LVEDD. For speckle tracking echocardiography, the myocardium was traced manually, and the EchoPac software then suggested an area of interest by delimiting the endocardium and the epicardium. The operator readjusted this area before the software calculated deformation. LV endocardial and epicardial global strain as well as LV longitudinal, LV early, and LV late diastolic strain rates were calculated from apical views. Negative values indicate fiber shortening, and positive values indicate fiber lengthening. If >1 segmen<sup>t</sup> was rejected, subjects were excluded from statistical analysis.
