Diamond is the first open source method for the quantitative assessment of four-dimensional segmental cardiac function in zebrafish embryos. Our method has a micrometer resolution that can quantify local cardiac mechanics in 3D space. No other current approach allows that.
Diamond unravels novel injury patterns in doxorubicin-induced cardiac toxicity. Therefore, we believe our method could be used as a platform for high throughput in vivo screening for chemotherapy-induced cardiac toxicity. We are currently working to adapt Diamond to the mammalian heart.
However, additional work is needed to take into account mammalian heart muscle fiber orientation and rotational and twisting motion. When attempting this method, it is very important that the user has a clear visualization of the anatomical structure of the zebrafish heart to correctly identify the horizontal and the vertical axes and to segment the ventricle. To reconstruct the 3D systolic and diastolic heart, begin by opening the folder created by the post-synchronization algorithm, then open the output folder.
Find the first systolic and diastolic phase and record the frame number. Then open the output by state folder and find the folders that have the same numbers as the recorded frame numbers. Convert the images in the folder to 3D TIF files and name them diastole.
tif and systole.tif. To perform segmentation of the ventricle, open the image analysis software by clicking file and open data. Then load diastole.
tif and systole. tif and enter the voxel size according to the image settings. Click the segmentation panel and manually segment the ventricle part of the heart using the built-in threshold tool.
Then remove the atrioventricular canal and the outflow tract in the segmented ventricle because this will affect the displacement analysis. After segmentation is completed, click the project panel, right-click the diastole.labels. tif and systole.labels.
tif tabs in the console and click export data as to save the data as 3D TIF files. Open prepimage_1. m in the programming environment, make sure that the folder in InPath on line five contains the original and segmented TIF files, and change slice on live four to the number of slices of the 3D TIF files.
After running the code, five new 3D TIF files and two new folders will be generated. Import all five 3D TIF files into the image analyzing software. Go to the multiplanar panel and choose diastole_200.
tif as the primary data. Align the x-axis with the vertical long axis of the ventricle and the z-axis with the horizontal long axis of the ventricle. Next, choose three random points from the oblique YZ plane in a counterclockwise manner and record their 3D position coordinates.
Repeat this process for systole_200.tif. Click the project panel and create a slice object for diastole_200. tif by right-clicking on diastole_200.
tif and searching for the slice object. Left-click the created slice object, check set plane in the properties panel options, choose three points in plane definition, and enter the previously recorded coordinates. Right-click diastole_200.
tif, search for resample transformed image and create the object. In the properties panel, choose slice as the reference and click apply which should generate an object named diastole_200.transformed. Next, right-click diastole_200.
transformed, search for resample and create the object. Choose voxel size as the mode and change it to x equals one, y equals one, and z equals one in the properties panel. Click apply then save the generated diastole_200.
resampled object as a 3D TIF file. Repeat this process for dialabel. tif, test.
tif, systole_200. tif, syslabel. tif, and test.
tif according to the manuscript directions. Import all six resampled files to ImageJ and select the slice of systole_200. resampled in which the atrioventricular canal is clearly visualized making sure to record the number of the slice.
Use the image transform rotate function to vertically position the atrioventricular canal. Apply the same rotation to all files then close all windows and save the changes. Next, move systole_200.
resampled, syslabel. resampled, and test2. resampled to the resample_sys folder.
Move diastole_200. resampled, dialabel. resampled, and test.
resampled to the resample_dia folder. Open divider_2_8_pieces. m and change InPath in line five to the image directory.
Change the variable middle in line 22 to the slice number where the atrioventricular canal is clearly visualized. Repeat this process for diastole_200. resampled on lines 394 and 411.
Run the code. When prompted, click once on the center of the ventricle and on the center of the atrioventricular canal. To register systolic and diastolic image matrices, open register_3.
m and change InPath in line four to the image folder path. Then open displacement_4. m and change InPath to the image folder path.
Run the program to generate a vector8. txt file with an eight by four matrix. Each row of the matrix contains the magnitudes of the x component, y component, z component, and the sum magnitude of the displacement vector of a specific segment of the ventricle.
These values can then be transferred to a spreadsheet. This protocol was used to uncover segmental heterogeneity of cardiac function and susceptibility to doxorubicin-induced myocardial injury in zebrafish. After a 24-hour doxorubicin treatment from three to four post-fertilization, displacement of ventricular segments was compared between control and treated groups.
Under control conditions, the basal segments one and six undergo the largest displacements and are the ones most susceptible to doxorubicin-induced cardiac injury. At six dpf, the average L2 norm of the segmental displacement vectors in the control fish ranged from 3.9 to 8%after normalization. The basal segments one and six recovered Diamond displacement to control levels suggesting segmental regeneration.
Meanwhile, a worsening in 2D basal strain from negative 53 to negative 38%was observed immediately after doxorubicin treatment followed by a return to control levels 48 hours later corroborating the Diamond displacement results. A parallel decrease and recovery of global ejection fraction or EF in response to treatment was also observed. Diamond was then applied during doxorubicin treatment and notch pathway modulation using the notch inhibitor DAPT and downstream effectors NICD and NRG1 mRNA.
The mRNA microinjection rescued the decrease in Diamond displacement and EF after acute chemotherapy-induced injury. Moreover, inhibition of the notch pathway after chemotherapy hindered the recovery of Diamond displacement of the basal segments and EF 48 hours after treatment, but the inhibition was rescued by the notch downstream effectors. Traditional ejection fraction is known to be an insensitive and delayed indicator of myocardial injury.
Diamond allows researchers to quantify focal cardiac function and assist the heterogeneity in myocardial injury in zebrafish embryos.
