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

Summary

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.

Abstract

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–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.

Introduction

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 injury^3,4. Tissue Doppler imaging provides ....

Protocol

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

1. Follow the housing, breeding, and embryo collection procedures as described in previously established husbandry and breeding practices. For details, see Messerschmidt et al.⁹.
2. Treat the collected embryos with 0.003% 1-phenyl-2-thiourea (PTU) in E3 medium 18 h postfertilization to maintain the transparency o....

Results

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 .......

Discussion

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 injury^1,4,12. 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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amira6	FEI		Image analyzing software
DAPT	Millipore Sigma	D5942-5MG
Doxorubicin hydrochloride	Millipore Sigma	D1515-10MG
Ethyl 3-aminobenzoate methanesulfonate	Millipore Sigma	E10521-10G	Tricaine
MATLAB	MathWorks		Programming environment
MATLAB Image Processing Toolbox	MathWorks		Image processing toolbox