Vevo 2100 machine

Echocardiography was performed on mice with the Vevo2100 machine (VisualSonics, Toronto) and a MS550D transducer 13 (link). Mice at the earliest ethically acceptable age (4 weeks) were anesthetized by isoflurane (4% for induction, 1.7% for experiment) and body temperature was monitored using a rectal temperature probe and controlled at 36-37 °C. B-mode loops of the left parasternal long-axis view of the heart were captured. Left ventricular (LV) volumes at end-diastole and end-systole were obtained, and ejection fraction (EF) was calculated. M-mode traces were obtained from short-axis 2-D view of the LV. LV dimensions at end-diastole and end-systole (LVDd, LVDs) were measured and fractional shortening (FS) was calculated [(LVDd - LVDs)/LVDd × 100%]. Thickness of anterior and posterior walls (AW, PW) of the LV was measured and LV mass was calculated as [(LVDd + AW + PW)³ - LVDd³] × 1.055. The right ventricle (RV) was visualized on the right parasternal long-axis view 13 (link). RV dimensions at end-systole and end-diastole were measured, and fractional shortening (RVFS) was calculated. All loops and images of three cardiac cycles were analyzed in a blinded fashion and the average was used.

Mice were sedated with 1% isoflurane with 21% oxygen delivered via nose cone at 1 l/min. ECG leads were placed for simultaneous ECG monitoring. Echocardiographic images were acquired on a Vevo 2100 machine using an MS400 transducer (VisualSonics Inc., Toronto, Canada). M-mode measurements at the midpapillary level of the left ventricle were performed to measure end-systolic and end-diastolic diameters and calculate stroke volume.^{39 (link)}

Left ventricular function was enumerated by transthoracic echocardiography, using a Vevo 2100 machine equipped with an MS550D transducer (Visual Sonics, Toronto, Canada) as described before (Veeranki et al. 2016). Briefly, the mice were anesthetized, using 3.5% isoflurane and depilated with Nair hair removal cream. Maintenance dose of isoflurane was 1.5% and heart rate was 450 ± 50 beats/min, while recording the cardiac parameters. Percentage ejection fraction (% EF) representing ventricular function, ventricular wall thickness, and chamber diameter were derived from the measurements during short‐axis M‐mode. LV fractional shortening (LVFS) was calculated according to the following equation: LVFS = ([LVEDD−LVESD]/LVEDD) × 100. Wherever possible, all of the physiological measurements were collected on the same period of the day to minimize variation.

All relevant ethical regulations for animal testing and research were followed. All procedures were performed following protocols approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee. Mice were maintained at 65–75°F with 40–60% humidity on a normal 12-hour light, 12-hour dark cycle. Myh7^YFP, R26^{fsCas9-P2A-GFP}, Rnf20^flox, and R26^fsTomato mice were described previously^{13 (link),20 (link),34 (link),54 (link)}. Echocardiography was performed on a VisualSonics Vevo 2100 machine with the Vevostrain software. Animals were awake during this procedure and held in a standard handgrip. The echocardiographer was blinded to genotype and treatment.