Three plane echocardiography to assess heart function in adult zebrafish.

(A) Overview of experimental setting and illustration of transducer positioning to image short axis view (SAX) (1), abdominal-cranial axis (ACX) for pulsed-wave Doppler (PWD) recordings (2), and long axis view (LAX) (3). (B) Lateral view of dorsally positioned adult zebrafish with three defined transducer positions illustrated. The red line indicates direction of ultrasound beam for PWD acquisition for cardiac inflow velocity imaging and the blue line indicates ultrasound beam direction for acquisition of cardiac outflow velocities. (C) Representative images of SAX view (1) and LAX view (2) acquisition (upper row). In the lower row the ventricle is outlined in red in (1) and (2). (D) PWD images derived from the ACX view (3) with the upper image showing representative signals attained from the AV-valve region with clearly visible positive A- and E- waves (in pink) and the bottom image displaying one representative V VTI PWD signal obtained from the bulbus arteriosus region. PWD, pulsed-wave Doppler.

Longitudinal echocardiographic evaluation of cardiac function after cryoinjury.

(A) Lateral brightfield (top) and fluorescent (bottom) images of hearts derived from sham operated transgenic zebrafish [Tg(myl7:GFP)^f1] and after cryoinjury at depicted time points. Dashed lines indicate injured myocardial area (i). (B) PWD recordings from sham (top) and cryoinjured zebrafish at 1dpi (bottom) demonstrating decreased A-wave and increased E-wave amplitudes indicative for diastolic dysfunction. (C) Representative SAX images from sham (upper row) and cryoinjured zebrafish at 1dpi (middle row) and at 30dpi (lower row) with end-diastolic dimensions illustrated in red and end-systolic dimensions in green. (D-I) Quantification of changes in (D) heart rate (HR), (E) fractional shortening (FS), (F) fractional area change (FAC), (G) ejection fraction (EF), (H) E/A ratio and (I) cardiac output (CO) at baseline and at indicated time points during regeneration after myocardial injury. Small number in (D) indicates number of animals analyzed (J) Speckle-tracking analysis of segmental displacement shows akinesia of injured (green, pink and light blue line) as compared to the non-injured segments (yellow, purple and dark blue line). The average curve of all segments is illustrated in black. For color coding of different segments see Fig 2B. Values are expressed as means ± SEM; a, atrium; i, injured area; ot, outflow tract; v, ventricle; AW, anterior wall; PW, posterior wall; *, p<0.05; unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test.

Advanced 2D wall motion measurement by speckle-tracking analysis after cryoinjury reveals segmental and regional motion and deformation disturbances.

(A) Absolute radial displacement of individual segments at indicated time points. The ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall (AW), 3 and 4 the apex (Ap), and 5 and 6 the posterior wall (PW) for subsequent displacement analysis. Small numbers indicate number of animals measured. (B) Average of radial displacement at indicated time points. (C) High resolution speckle-tracking analysis of radial (upper row) and longitudinal (lower row) displacement. The top region displays displacement of the AW, the central region of the Ap and the lower region of the PW. Absolute values are color coded with high values in light red and low values in light blue as indicated. Time scale and color coding bar as indicated. The pink line at 4dpi depicts injured area (MI). (D) 3D reconstruction of regional displacement at indicated time points enables identification of akinesis of injured AW at 4dpi and residual wall motion deficiencies at 60dpi, respectively. The lower right image shows a schematic illustration of the 3D-reconstrations. The u-shaped pink lines indicate consecutive systoles; t and the arrow below indicate the time progress and MI the infarcted area, also indicated by the bold pink line. (E) Modified AFOG-staining (myocardium in red, connective tissue and fibrotic areas in blue) stained sections of a sham operated control heart and at 120 and 180dpi. AW is to the right, PW to the left. Boxed area of the cryo-injured region is shown in higher magnification in the lower left corner of its respective overview picture. At 120dpi residual fibrotic deposition (arrows) together with a thickening of the compact myocardial layer (*) can be detected. Residues of fibrosis are still detectable at 180dpi. Note that the thickening of the compact myocardial layer (*) extends over a great segment of the AW. Ap, apex; A, atrium, AVV, atrio-ventricular valve; AW, anterior wall; B, bulbus arteriosus; MI, myocardial injury; PW, posterior wall; V, ventricle; VBV, ventriculo-bulbar valve. Values are expressed as means ± SEM; *, p<0.05; **, p<0.01, unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test