Vevo 3100

The cardiac function of mice was evaluated by transthoracic echocardiography [22 (link)] (Vevo 3100 imaging system, Vevo 3100, FUJIFILM VisualSonics lnc., Toronto, ON, Canada). Two experienced echo-cardiologists performed the M-mode echocardiography. At the papillary level, the echocardiography parameters, including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD), were recorded from the mice anesthetized with isoflurane. All the data were measured in five consecutive cardiac cycles.

Rats were mildly anesthetized using sodium pentobarbital. After removing the hairs on the chest with depilatory paste, the rat was placed on an experimental animal plate with a heating function. Then the electrocardiogram metal electrode on the animal plate was coated with a conductive paste. Next, the chest of each rat was covered with an ultrasonic coupling agent. M-mode echocardiography was performed to measure cardiac function using a Vevo3100 instrument with a 38 MHz transducer (Visual Sonics). The cardiac parameters including left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic diameter (LVIDd), anterior wall thickness (AWT), the ratio of mitral peak velocity during early diastole to atrial contraction (E/A ratio), ejection fraction (EF), and fractional shortening (FS) were measured and calculated.

All measurements and assessments were performed using the Visual Sonics Echo system (Vevo 3100, VisualSonics, Inc., Toronto, Canada) and the 15–30 MHz (MX250) linear transducer (VisualSonics). Animals were placed in the echocardiography room for at least 30 min before examination. Rats were maintained unconscious using 2% isoflurane and placed in a supine position on a heating platform. The chest was shaved and ultrasonic gel applied to the thoracic area to allow maximal visibility of the heart chambers. The ultrasonic probe was placed on the chest along the long-axis of the left ventricle and adjusted to obtain clear two-dimensional B-mode and M-mode parasternal long axis images. Five minutes was allowed for each animal to stabilize in that position before acquiring any measurements. Heart rate was maintained constant throughout the procedure (350–400 beats/min). Note that only a subset of rats in the study were used to measure cardiac function. The number of rats used for each experiment are presented in the figure legend.

After 3 weeks of Ang-II infusion, the mice were anesthetized with 1.0% inhaled isoflurane and cardiac echocardiography was performed using an ultra-high-resolution small animal ultrasound imaging system (VisualSonics Vevo 3100) to evaluate the structural parameters and cardiac function. The data were analyzed from three consecutive cardiac cycles of each measurement. Several indicators, including left ventricular anterior wall thickness (LVAW), LV ejection fraction (EF, %), and LV fractional shortening (FS, %), were collected. Subsequently, the mice were euthanized to remove hearts, and body weight (BW) and heart weight (HW) were obtained to calculate the HW:BW ratio. Furthermore, the widest part of the middle ventricular tissue of hearts was collected and fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into sections of 5 μm thickness. Then the sections were stained with hematoxylin and eosin (H&E, Sigma) to observe the general morphology. The sections were incubated with wheat germ agglutinin (WGA, Sigma) to evaluate the cross‐sectional areas of cardiomyocytes.