Visualsonics vevo 2100

Mice were preconditioned by chest hair removal with a topical depilatory (FujiFilm VisualSonics), anaesthetized with 1.5–2.5% isoflurane administered via inhalation, and maintained in a supine position on a dedicated animal handling platform with limbs attached for electrocardiogram gating during imaging. Body temperature was kept constant by feeding the signal of a rectal probe back to a heating pad, while heart and respiratory rates were continuously monitored. Transthoracic echocardiography was performed using a high frequency ultrasound system dedicated to small animal imaging (VisualSonics Vevo 3100, FujiFilm VisualSonics) using a MX 400 linear array transducer (20–46 MHz). Experiments for Supplementary Fig. 4d–f were performed using a high frequency ultrasound system dedicated to small animal imaging (VisualSonics Vevo 2100, FujiFilm VisualSonics) and a MS 400 linear array transducer (18–38 MHz). M-mode recording was performed at the midventricular level. Percent LV fractional shortening (%FS) was calculated from M-mode measurements. All images were analyzed using dedicated software (Vevo Labo version 5.7.1 or Vevo 2100 version 1.4).

Histology slides were imaged using Aperio ScanScope and analyzed using ImageScope software. Fluorescent labeled immunostained slides were taken with a Nikon microscope. In consideration to the 3D and clarified nature of the samples, advanced multiphoton microscopy, at a core facility (Center for Cellular Imaging, Sweden), was utilized to perform volume imaging using a LSM 710 NLO Zeiss microscope coupled to a Mai Tai Deepsee or InSight laser (Spectra-Physics) with integrated pump laser tuned at 860 nm^{26 (link)}. Multiphoton-excited autofluorescence and second harmonic generation (SHG) is advantageous for volumetric imaging to investigate cell-cell and cell-matrix interactions^{27 (link),28 (link)}. The fine structures and organization of elastin is obtained with autofluorescence while the anisotropic collagen fibers generate SHG signals. Due to the limited autofluorescence of the cardiac fibroblasts, Hoechst staining was used to highlight the cells. The success of 4-Flow hearts is the ventricle pacing and competence of the aortic valve which can be visualized noninvasively and under sterile conditions with ultrasound imaging was performed using the FujiFilm VisualSonics VEVO 2100 with the 40 MHz probe. Images were taken at 20 frames per second and compiled into a video. Still images of the ultrasound video were analyzed using ImageJ.

Mice were preconditioned by chest hair removal with a topical depilatory (FujiFilm VisualSonics, Toronto, Canada), anaesthetized with 1.5–2.5% isoflurane administered via inhalation, and maintained in a supine position on a dedicated animal handling platform with limbs attached for electrocardiogram gating during imaging. Body temperature was kept constant by feeding the signal of a rectal probe back to a heating pad, while heart and respiratory rates were continuously monitored. Transthoracic echocardiography was performed using a high frequency ultrasound system dedicated to small animal imaging (VisualSonics Vevo 2100, FujiFilm VisualSonics, Toronto, Canada) using a MS 400 linear array transducer (18–38 MHz). M-mode recording was performed at the midventricular level. All images were analyzed using dedicated software (Vevo 2100 version 1.4). LV wall thickness and internal cavity diameters at diastole (LVID;d) and systole (LVID;s) were measured. Per cent LV fractional shortening (%FS) was calculated from M-mode measurements. All procedures were performed under double-blind conditions with regard to genotype or treatment.

Mice were preconditioned by chest hair removal using a topical depilatory (FujiFilm VisualSonics, Toronto, Canada), anesthetized with 1.5–2.5% isoflurane administered via inhalation, and maintained in a supine position on a platform with limbs attached for electrocardiogram gating during imaging. Body temperature was kept constant by feeding the signal of a rectal probe back to a heating pad, while heart and respiratory rates were continuously monitored. Transthoracic echocardiography was performed using a high-frequency ultrasound system for small animal imaging (VisualSonics Vevo 2100, FujiFilm VisualSonics, Toronto, Canada) using an MS 400 linear array transducer (18–38 MHz). M-mode recording was performed at the midventricular level. All images were analyzed using Vevo 2100 version 1.4 software. Left ventricle wall thickness and internal cavity diameters at diastole and systole were measured. Left ventricle volumes in diastolic phases (LV Vol d) and systolic phases (LV Vol s) were measured. The ejection fraction (%) was calculated as [(LV Vol d) - (LV Vol s)] (LV Vol d)⁻¹ × 100. All procedures were performed under double-blind conditions with regard to genotype or treatment.