Name: Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish (Video) | JoVE Journal
Uploaded: 2020-02-06T14:00:00.000+00:00
Duration: 9 min 15 s
Description: Discover the DIAMOND method for assessing segmental cardiac function in embryonic zebrafish with micrometer resolution. This open-source technique enables 3D quantification of local cardiac mechanics for high-throughput drug toxicity screening.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.</li>
	<li>Treat the collected embryos with 0.003% 1-phenyl-2-thiourea (PTU) in E3 medium 18 h postfertilization to maintain the transparency of the embryos for LSFM imaging.</li>
</ol>
2. Doxorubicin treatment to induce cardiac injury
<ol>
	<li>At 3 days postfertilization (dpf), treat the embryos with doxorubicin at a concentration of 10 μM in E3 fish water medium. After a 24 h treatment to 4 dpf, replace the doxorubicin medium with fresh E3 medium. 
	CAUTION: Doxorubicin is a chemotherapy medication. Appropriate personal protective equipment (PPE) is required and the waste should be disposed of in biohazard waste containers.</li>
</ol>
3. Notch pathway modulation
<ol>
	<li>Treat zebrafish embryos with the Notch pathway inhibitor (2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT) at a concentration of 10 μM in E3 fish water medium from 3–6 dpf.</li>
	<li>Microinject the Notch downstream effectors Notch intracellular domain (NICD) and Neuregulin-1 (Nrg-1) mRNA at concentrations of 10 pg/nL and 5 pg/nL, respectively, into the 1-cell stage zebrafish embryos8,10. 
	NOTE: The microinjection is performed under a microscope with the support of an air pump to accurately control the volume injected. The mRNA microinjection into the cell is done when the fertilized egg is at the first cell stage. For details on the preparation and sequence of the mRNAs, see Chen et al.8. For details on the microinjection and preparation of injection needles, see Rosen et al.10.</li>
</ol>
4. LSFM imaging and post-imaging synchronization
<ol>
	<li>For the LSFM imaging techniques and post-imaging synchronization algorithm, see details in previous publications9,11. 
	NOTE: Briefly, our system utilizes a continuous-wave laser as the illumination source to image all transgenic zebrafish lines. The detection module is composed of two scientific complementary metal-oxide semiconductor (sCMOS) cameras and two sets of filters for dual-channel imaging. The detection module is perpendicularly installed to the illumination plane. Each LSFM frame is acquired within a 20 msec exposure time, while the resolving power in cross section is ~0.65 μm and the step size between consecutive frames is ~2 μm. A 589 nm laser was used to excite mCherry fluorescent signals.</li>
</ol>
5. Reconstruction of the 3D systolic and diastolic heart
<ol>
	<li>Open the folder created by the post synchronization algorithm, then open the "Output" folder. Select the middle plane of the heart and load the entire folder into ImageJ. Find the first diastolic and systolic phase and record the frame number.</li>
	<li>Open the "Output/By State" folder and find the folders that have the same numbers as the frame numbers just recorded. Convert the images in the folder into 3D TIFF (tagged image file format) files and name them "diastole.tif" and "systole.tif".</li>
</ol>
6. Segmentation of the ventricle
<ol>
	<li>Open the image analyzing software (see Table of Materials). Click File | Open data, and load "diastole.tif" and "systole.tif". Enter the voxel size according to the imaging settings. 
	NOTE: For the LSFM system used, the typical voxel size is 0.65 µm x 0.65 µm x 2 µm.</li>
	<li>Click the "SEGMENTATION" panel and manually segment out the ventricle part of the heart. The built-in "Threshold" tool that can select all the regions above a certain intensity can facilitate this process. The ventricle is the thicker chamber with a stronger fluorescence. 
	NOTE: Make sure to remove the atrioventricular canal and the outflow tract in the segmented ventricle, because this affects the displacement analysis.</li>
	<li>After the segmentation is done, 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 TIFF files.</li>
</ol>
7. Creation of rectangular parallelepipeds for image registration
<ol>
	<li>Run "prepImage_1.m" in the programming environment (see Table of Materials). Open "prepImage_1.m", "ImPath" in line 5 so the folder contains the original and segmented TIFF files, and change "slice" in line 4 to the number of slices of the 3D tif files.</li>
	<li>After running the code, it will generate five new 3D TIFF files ("test.tif", "diastole_200.tif", "systole_200.tif", "diaLabel.tif", and "sysLabel200.tif") as well as two new folders ("resample_dia" and "resample_sys").</li>
</ol>
8. Resample systolic and diastolic 3D hearts along the short axis plane
<ol>
	<li>Import all five 3D TIFF files into the image analyzing software (see Table of Materials). 
	NOTE: The voxel size is unchanged.</li>
	<li>Go to the MULTIPLANAR panel. Choose "diastole_200.tif" as the primary data. Align the X-axis (the green line in the XY plane) with the vertical long axis of the ventricle, and align the Z-axis (the red line in the YZ plane) with the horizontal long axis of the ventricle. 
	NOTE: The vertical long axis is determined by finding the longest axis connecting the apex and the outflow tract in the XY plane, and the horizontal long axis is determined by finding the longest axis connecting the apex and the outflow tract in the YZ plane. Rotate the axis by placing the cursor at the end of the axis.</li>
	<li>Choose three random points from the oblique YZ plane (the short axis plane) in a counterclockwise manner and record their 3D position coordinates. 
	NOTE: Make sure the points are chosen in a counterclockwise manner.</li>
	<li>Repeat steps 8.2 and 8.3 for "systole_200.tif".</li>
	<li>Click the "PROJECT" panel. Create a "Slice" object for "diastole_200.tif" by right clicking on "diastole_200.tif" and searching for "Slice" object. Left click the Slice object just created, and in the Properties panel | Options, check "Set Plane" and choose three points in "Plane Definition". Enter the coordinates of the three points from step 7.3.</li>
	<li>Repeat step 8.5 for "systole_200.tif". 
	NOTE: The slice object created should have the name "Slice 2".</li>
	<li>Right click "diastole_200.tif" and search for "Resample Transformed Image" and create the object. In the Properties panel, choose "Slice" as the "Reference" and click Apply. This should generate an object named "diastole_200.transformed".</li>
	<li>Right click "diastole_200.transformed" and search for "Resample" and create the object. Choose "Voxel Size" as the "Mode" and change "Voxel Size" to be x = 1, y = 1, and z = 1 in the Properties panel.</li>
	<li>Click "Apply". This should generate an object named "diastole_200.resampled". Right click "diastole_200.resampled" and save it as a 3D TIFF file.</li>
	<li>Repeat the same step for "diaLabel.tif" and "test.tif". Save "diaLabel.resampled" and "test.resampled" as 3D TIFF files. Repeat the same step for "systole_200.tif", "sysLabel.tif", and "test.tif" using "Slice 2" as a reference, and save "systole_200.resampled", "sysLable.resampled", and "test2.resampled" as 3D TIFF files. 
	NOTE: Make sure there are a total of six TIFF files saved in this step.</li>
</ol>
9. Division of the resampled heart
<ol>
	<li>Import all six resampled files from step 8 to ImageJ. Select a slice of "systole_200.resampled" in which the atrioventricular canal is clearly visualized. Record the number of the slice.
	<ol>
		<li>Use the "Image | Transform | Rotate" function of ImageJ so that the atrioventricular canal is vertical. Apply the same rotation to all files. Close all windows and save all changes.</li>
		<li>Move "diastole_200.resampled", "diaLabel.resampled", and "test.resampled" to the "resample_dia" folder, and move "systole_200.resampled", "sysLable.resampled", and "test2.resampled" to the "resample_sys" folder.</li>
	</ol>
	</li>
	<li>Open "divider_2_8_pieces.m". Change "ImPath" in line 5 and "ImPath" in line 395 to the image directory. Change the variable "Middle" in line 22 and line 411 to the slice numbers where the atrioventricular canal is clearly visualized in "systole_200.resampled" and "diastole_200.resampled".</li>
	<li>Run the code, and in the prompted windows click once at the center of the ventricle and click once at the center of the atrioventricular canal. This needs to be done twice for both systole and diastole images.</li>
</ol>
10. Registration of systolic and diastolic image matrices
<ol>
	<li>Open "register_3.m" and change "ImPath" in line 4 to the image folder path. It might take 5–20 min to run this code depending on the computation power of the system. 
	NOTE: The artificially created rectangular parallelepipeds in step 7 are used for 3D rigid registration that preserves the distance between two points and angles subscribed by three points. When the end-diastole rectangular parallelepiped (red) is registered to the end-systole rectangular parallelepiped (green), the ensuing discrepant 3D location permits the derivation of a unique matrix of rigid transformation consisting of rotation and translation from the end-diastole matrix to the end-systole matrix (Figure 1H). We perform the registration and regularized energy minimization to denoise the matrix after the transformation using an image processing toolbox (see Table of Materials). For a detailed mathematical description, please see Chen et al.8.</li>
</ol>
11. Output of the displacement vectors
<ol>
	<li>Open "displacement_4.m" and change "ImPath" in line 4 to the image folder path.</li>
	<li>Run "displacement_4.m", which generates a "vector8.txt" file in the "vectors" folder. Once the "vector8.txt" file is open, there will be an 8 x 4 matrix. Each row of the matrix has four numbers, which are 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. 
	NOTE: The displacement vector is obtained by calculating the displacement of the mass centroid of each segment in 3D space. We calculate the 3D mass centroid (PS and PD) coordinates <img alt="Static equilibrium concept, shown by ΣFx=0 equation, with supportive diagram and equilibrium analysis." src="/files/ftp_upload/60547/60547eqck.jpg" data-altupdatedat="1788250015000" data-alt="Equation CK"> (where k indicates the X, Y, or Z coordinate, respectively) of each segment (I-VI) in the segmentation dataset from systole to diastole (Figure 1J). We define the mass centroid <img alt="Static equilibrium concept, shown by ΣFx=0 equation, with supportive diagram and equilibrium analysis." src="/files/ftp_upload/60547/60547eqck.jpg" data-altupdatedat="1788250015000" data-alt="Equation CK"> in 3D space as follows: 
	<img alt="Average concentration formula, Σcρ(x,y,z), summation method, chemistry equation, educational usage." src="/files/ftp_upload/60547/60547eq1.jpg" data-altupdatedat="1788250015000" data-alt="Equation 1"> 
	where Cx = X, Cy = Y, and Cz = Z, Mi = the mass of each segment (I ≤ i ≤ VI), m = the number of voxels of each segment, and ρ = the density function as the segmented region is 1 whereas the rest is 0. The L2-norm of the sub-displacement vectors along the X-, Y-, and Z-axes and the sum displacement vector are calculated during the cardiac cycle. There are a total of eight rows in the matrix. The first row and the eighth row contain the atrioventricular canal and are thus ignored in our analysis. Segments I to VI are represented by the second row to the seventh row.</li>
</ol>

<ol><li>Ewer, M. S., Ewer, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiotoxicity+of+anticancer+treatments%5BTitle%5D)+AND+%22Nature+Reviews+Cardiology%22%5BJournal%5D)">Cardiotoxicity of anticancer treatments.</a> Nature Reviews Cardiology. 12 (9), 547-558 (2015).</li>
<li>Thavendiranathan, P., Wintersperger Bernd, J., Scott, F. D., Thomas D, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+MRI+in+the+Assessment+of+Cardiac+Injury+and+Toxicity+From+Cancer+Chemotherapy%5BTitle%5D)+AND+%22Circulation%3A+Cardiovascular+Imaging%22%5BJournal%5D)">Cardiac MRI in the Assessment of Cardiac Injury and Toxicity From Cancer Chemotherapy.</a> Circulation: Cardiovascular Imaging. 6 (6), 1080-1091 (2013).</li>
<li>Mickoleit, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+reconstruction+of+the+beating+zebrafish+heart%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">High-resolution reconstruction of the beating zebrafish heart.</a> Nature Methods. 11 (9), 919-922 (2014).</li>
<li>Thavendiranathan, P., et al. Use of Myocardial Strain Imaging by Echocardiography for the Early Detection of Cardiotoxicity in Patients During and After Cancer Chemotherapy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aThavendiranathan%2C+P+%22A+Systematic+Review%22">A Systematic Review</a>. 63 (25), Part A 2751-2768 (2014).</li>
<li>Collier, P., Phelan, D., Klein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Test+in+Context%3A+Myocardial+Strain+Measured+by+Speckle-Tracking+Echocardiography%5BTitle%5D)+AND+%22Journal+of+the+American+College+of+Cardiology%22%5BJournal%5D)">A Test in Context: Myocardial Strain Measured by Speckle-Tracking Echocardiography.</a> Journal of the American College of Cardiology. 69 (8), 1043-1056 (2017).</li>
<li>Hanekom, L., Cho, G. Y., Leano, R., Jeffriess, L., Marwick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+two-dimensional+speckle+and+tissue+Doppler+strain+measurement+during+dobutamine+stress+echocardiography%3A+an+angiographic+correlation%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">Comparison of two-dimensional speckle and tissue Doppler strain measurement during dobutamine stress echocardiography: an angiographic correlation.</a> European Heart Journal. 28 (14), 1765-1772 (2007).</li>
<li>Poss, K. D., Wilson, L. G., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+regeneration+in+zebrafish%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Heart regeneration in zebrafish.</a> Science. 298 (5601), 2188-2190 (2002).</li>
<li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Displacement+analysis+of+myocardial+mechanical+deformation+%28DIAMOND%29+reveals+segmental+susceptibility+to+doxorubicin-induced+injury+and+regeneration%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Displacement analysis of myocardial mechanical deformation (DIAMOND) reveals segmental susceptibility to doxorubicin-induced injury and regeneration.</a> JCI Insight. 4 (8), e125362(2019).</li>
<li>Messerschmidt, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light-sheet+Fluorescence+Microscopy+to+Capture+4-Dimensional+Images+of+the+Effects+of+Modulating+Shear+Stress+on+the+Developing+Zebrafish+Heart%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart.</a> Journal of Visualized Experiments. (138), e57763(2018).</li>
<li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> Journal of Visualized Experiments. (25), e1115(2009).</li>
<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((4-Dimensional+light-sheet+microscopy+to+elucidate+shear+stress+modulation+of+cardiac+trabeculation%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.</a> The Journal of Clinical Investigation. 126 (5), 1679-1690 (2016).</li>
<li>Lenneman, C. G., Sawyer, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardio-Oncology%3A+An+Update+on+Cardiotoxicity+of+Cancer-Related+Treatment%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">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/pubmed?term=((Assessment+of+Myocardial+Mechanics+Using+Speckle+Tracking+Echocardiography%3A+Fundamentals+and+Clinical+Applications%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Echocardiography%22%5BJournal%5D)">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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+pitfalls+of+strain+rate+imaging%3A+angle+dependency%5BTitle%5D)+AND+%22Biomedical+Sciences+Instrumentation%22%5BJournal%5D)">Potential pitfalls of strain rate imaging: angle dependency.</a> Biomedical Sciences Instrumentation. 36, 197-202 (2000).</li>
<li>Seo, Y., Ishizu, T., Aonuma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+Status+of+3Dimensional+Speckle+Tracking+Echocardiography%3A+A+Review+from+Our+Experiences%5BTitle%5D)+AND+%22Journal+of+Cardiovascular+Ultrasound%22%5BJournal%5D)">Current Status of 3Dimensional Speckle Tracking Echocardiography: A Review from Our Experiences.</a> Journal of Cardiovascular Ultrasound. 22 (2), 49-57 (2014).</li>
<li>Amzulescu, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improvements+of+Myocardial+Deformation+Assessment+by+Three-Dimensional+Speckle-Tracking+versus+Two-Dimensional+Speckle-Tracking+Revealed+by+Cardiac+Magnetic+Resonance+Tagging%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Echocardiography%22%5BJournal%5D)">Improvements of Myocardial Deformation Assessment by Three-Dimensional Speckle-Tracking versus Two-Dimensional Speckle-Tracking Revealed by Cardiac Magnetic Resonance Tagging.</a> Journal of the American Society of Echocardiography. 31 (9), 1021-1033 (2018).</li>
<li>Wolterink, J. M., Leiner, T., Viergever, M. A., Išgum, I. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWolterink%2C+JM+%22Reconstruction%2C+Segmentation%2C+and+Analysis+of+Medical+Images%22">Reconstruction, Segmentation, and Analysis of Medical Images</a>. Zuluaga, M. A., et al. , Springer International Publishing. 95-102 (2016).</li>
<li>Avendi, M. R., Kheradvar, A., Jafarkhani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+combined+deep-learning+and+deformable-model+approach+to+fully+automatic+segmentation+of+the+left+ventricle+in+cardiac+MRI%5BTitle%5D)+AND+%22Medical+Image+Analysis%22%5BJournal%5D)">A combined deep-learning and deformable-model approach to fully automatic segmentation of the left ventricle in cardiac MRI.</a> Medical Image Analysis. 30, 108-119 (2016).</li>
<li>Packard, R. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+Segmentation+of+Light-Sheet+Fluorescent+Imaging+to+Characterize+Experimental+Doxorubicin-Induced+Cardiac+Injury+and+Repair%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Automated Segmentation of Light-Sheet Fluorescent Imaging to Characterize Experimental Doxorubicin-Induced Cardiac Injury and Repair.</a> Scientific Reports. 7 (1), 8603(2017).</li>
<li>Jay Kuo, C. C., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+data-driven+Saak+transform%5BTitle%5D)+AND+%22Journal+of+Visual+Communication+and+Image+Representation%22%5BJournal%5D)">On data-driven Saak transform.</a> Journal of Visual Communication and Image Representation. 50, 237-246 (2018).</li>
<li>Natarajan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement+Receptor+C5aR1+Plays+an+Evolutionarily+Conserved+Role+in+Successful+Cardiac+Regeneration%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration.</a> Circulation. 137 (20), 2152-2165 (2018).</li>
<li>Zhukov, L., Barr, A. H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aZhukov%2C+L+%22IEEE+Visualization+VIS+2003%22">IEEE Visualization VIS 2003</a>. , 597-602 (2003).</li>
<li>Nielles-Vallespin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+diffusion+tensor+MRI+of+the+human+heart%3A+Reproducibility+of+breath-hold+and+navigator-based+approaches%5BTitle%5D)+AND+%22Magnetic+Resonance+in+Medicine%22%5BJournal%5D)">In vivo diffusion tensor MRI of the human heart: Reproducibility of breath-hold and navigator-based approaches.</a> Magnetic Resonance in Medicine. 70 (2), 454-465 (2013).</li>
</ol>

The authors have declared that no conflict of interest exists.

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.

&#160; &#160;

<table><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

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 ...

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.

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

displacement-analysis-myocardial-mechanical-deformation-diamond

Research

JoVE Journal

Developmental Biology

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A rapid way to conduct immunostaining of zebrafish embryonic heart is described.  Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunostaining of Dissected Zebrafish Embryonic Heart 

This video describes a technique of isolating the heart from a zebrafish at multiple developmental stages to study cardiac development, maturation, and ageing.

Heart Dissection: A Method to Observe Zebrafish Cardiac Development

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

This protocol describes a low-cost light microscopy method to assess cardiac morphology and function in zebrafish embryos, enabling reproducible evaluation of developmental cardiotoxicity without the need for advanced imaging systems.

Functional Cardiac Imaging in Zebrafish Embryos Using Standard Microscopy and Video Analysis: Applications in Environmental and Biomedical Research

Discover the DIAMOND method for assessing segmental cardiac function in embryonic zebrafish with micrometer resolution. This open-source technique enables 3D quantification of local cardiac mechanics for high-throughput drug toxicity screening.

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

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

Explore More Videos