Vevolab analysis software

Prior to echocardiography, mice and rats were anesthetized with isoflurane. Unconscious mice and rats were weighed and secured in a supine position on a temperature-controlled restraining board. Anesthesia was maintained with 1–2% isoflurane in oxygen delivered through a nose cone. All hair in the thoracic region was removed using a depilatory agent, and the area was cleaned with water. Echocardiographic images were obtained with a Vevo 3100 system (Visualsonics, Inc.) as previously described [10 (link),12 (link),13 (link)]. Briefly, parasternal long and short axis views were obtained with both M-mode and two-dimensional echocardiography. LV dimensions (LV end diastolic diameter, LVEDD and LV end systolic diameter, LVESD) were measured perpendicular to the long axis of the ventricle at the mid-chordal level on three consecutive cycles and averaged by two independent observers (J.S. and M.A.) in a blinded fashion. Fractional shortening and LV ejection fraction were accordingly calculated. Advanced cardiac analysis (regional and global cardiac measurements) was assessed by speckle-tracking echocardiography (Vevo LAB analysis software; VisualSonics). Image analysis was performed according to manufacturer’s instructions as previously reported [10 (link),13 (link)]. The n value for each experimental group is specified in the relative figure legends.

It is challenging to obtain non-invasive images of the hearts of animals, such as mice, due to their small size and very fast heart rate. Therefore, echocardiography was performed once the mice were anesthetized with 2.5% isoflurane inhalation to reduce stress and to maintain a heart rate ranging from 475 to 525 bpm [29 (link)], which minimizes variations in measurements. We used a 21 MHz ultrasound probe and a Vevo^® 2100 System (Visual Sonics, Toronto, ON, Canada). All the ultrasound measurements were recorded in our small animal imaging facility at the CRCHU. To limit analysis bias, all the images were acquired and analyzed by a “blind” experimenter using the papillary muscles as a reference point [30 (link)]. High-quality images with an adequate delineation of the endocardial borders were used for hemodynamic analyses. The small-axis parasternal view using M-mode imaging was recorded to measure the anterior and the posterior left ventricular (LV) wall thickness and the LV internal diameter (Tables S1 and S2). The mass of the LV, the ejection fraction (EF), and the fractional shortening (FS) were calculated using the built-in Vevo LAB analysis software (Visual Sonics, software version 5.6, Toronto, ON, Canada).

Mice were anesthetized (1.5–3% isoflurane (N01A B06 (Isoflurane), Baxter AS, Norway) and placed on a heated imaging platform (Vevo 3100, VisualSonics, Toronto, Canada). Body temperature, heart- and respiration rate were constantly monitored. Mouse hearts were imaged using a frequency (40 MHz) linear array transducer (MX550D, VisualSonics, Toronto, Canada) on an echocardiography machine (Vevo 3100, VisualSonics, Toronto, Canada). All echocardiography acquisitions and image analyses were performed and evaluated by blinded assessment. VevoLAB analysis software was used for the analysis (VisualSonics, Toronto, Canada).