Name: displacement analysis of myocardial mechanical deformation (diamond) reveals segmental heterogeneity of cardiac function in embryonic zebrafish
Uploaded: 2020-02-06T14:00:00
Description: Watch this Scientific Journal Video about Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish at JoVE.com

The present work was funded by American Heart Association grants 16SDG30910007 and 18CDA34110338, and by National Institutes of Health grants HL083015, HL111437, HL118650, and HL129727.

All methods described here have been approved by the UCLA Institutional Animal Care and Use Committee (IACUC), and experiments were performed in compliance with protocols approved by the UCLA Office of Animal Research.
1. Breeding Tg(cmlc2:mCherry) zebrafish and collection of embryos
<ol>
	<li>Follow the housing, breeding, and embryo collection procedures as described in previously established husbandry and breeding practices. For details, see Messerschmidt et al.9...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardio-Oncology:+An+Update+on+Cardiotoxicity+of+Cancer-Related+Treatment.">Cardio-Oncology: An Update on Cardiotoxicity of Cancer-Related Treatment.</a> Circulation Research. 118 (6), 1008-1020 (2016).</li><li>Geyer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Myocardial+Mechanics+Using+Speckle+Tracking+Echocardiography:+Fundamentals+and+Clinical+Applications.">Assessment of Myocardial Mechanics Using Speckle Tracking Echocardiography: Fundamentals and Clinical Applications.</a> Journal of the American Society of Echocardiography. 23 (4), 351-369 (2010).</li><li>Castro, P. L., Greenberg, N. L., Drinko, J., Garcia, M. J., Thomas, J. 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A rigorous strategy for quantification of segmental myocardial function is critical to assess cardiac mechanics beyond traditional EF, known to be an insensitive and delayed indicator of myocardial injury1,4,12. Hence, there has been a growing interest in markers of early myocardial changes, and a growing body of literature supports myocardial deformation parameters as an early indicator to forecast ventricular dysfunction<sup c...

Chemotherapy-induced cardiac toxicity and ensuing heart failure are one of the main reasons for chemotherapy discontinuation1. Therefore, cardiac functional assessment plays a crucial role in the identification of cardiac toxicity and, more importantly, in the prediction of early cardiac injury following chemotherapy2. However, current approaches for cardiac functional assessment encounter limitations. Methods such as left ventricular ejection fraction (LVEF) provide only global and often delayed cardiac mechanics after injury3,4. Tissue Doppler imaging provides segmental myocardial deformation information but suffers from significant intraobserver and interobserver variability, in part due to ultrasound beam angle dependency5. Two-dimensional (2D) speckle tracking utilizes the B-mode of echocardiography, which theoretically eliminates the angle dependency, but its accuracy is limited by out-of-plane motion6. Therefore, a rigorous approach for quantifying segmental cardiac function is lacking in both research and clinical settings.
In this context, we developed a 4D quantification method for the analysis of segmental cardiac function that we named displacement analysis of myocardial mechanical deformation (DIAMOND), to determine the displacement vectors of myocardial mass centroids in 3D space. We applied DIAMOND for the in vivo assessment of cardiac function and doxorubicin-induced cardiac toxicity with zebrafish (Danio rerio) as the animal model, chosen due to their regenerating myocardium and highly conserved developmental genes7. We further compared segmental DIAMOND displacement with global ejection fraction (EF) determination and 2D strain following doxorubicin treatment. By integrating DIAMOND displacement with 4D light-sheet fluorescent microscopy (LSFM) acquired rendering of embryonic zebrafish hearts, DIAMOND shows that the basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to acute doxorubicin cardiac injury8.

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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amira6</td>
			<td>FEI</td>
			<td></td>
			<td>Image analyzing software</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Millipore Sigma</td>
			<td>D5942-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>D1515-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Millipore Sigma</td>
			<td>E10521-10G</td>
			<td>Tricaine</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Programming environment</td>
		</tr>
		<tr>
			<td>MATLAB Image Processing Toolbox</td>
			<td>MathWorks</td>
			<td></td>
			<td>Image processing toolbox</td>
		</tr>
	</tbody>
</table>

displacement analysis of myocardial mechanical deformation (diamond) reveals segmental heterogeneity of cardiac function in embryonic zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

The process by which DIAMOND was developed to assess 3D segmental cardiac function is presented in Figure 1. Following LSFM image acquisition and reconstruction in 3D of the embryonic zebrafish heart (Figure 1A), the true short axis plane was determined as the plane perpendicular to the vertical and horizontal long axes, both of which are determined in a multiplane viewer (Figure 1B). The heart was ...

Watch this Scientific Journal Video about Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish at JoVE.com

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

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.

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