心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性
Summary
该协议的目的是详细说明一种在生理和病理条件下评估胚胎斑马鱼分段心脏功能的新方法。
Abstract
斑马鱼越来越多地被用作心肌病和再生的模范生物体。目前评估心脏功能的方法无法可靠地检测分段力学，在斑马鱼中不容易实现。在这里，我们提出了一种半自动化的开源方法，用于四维（4D）分段心脏功能的定量评估：心肌机械变形（DIAMOND）的位移分析。转基因胚胎斑马鱼在体内使用具有4D心脏运动同步的光片荧光显微镜系统进行成像。获得 3D 数字心脏在端端和端端重建，心室被手动分割成二进制数据集。然后，心脏被重新定向，沿着真正的短轴对同热带重新采样，心室沿着短轴均匀地分成八个部分（I+VIII）。由于在端缩和端斜面处的不同重采样平面和矩阵，应用了变换矩阵进行图像配准，以恢复重新采样收缩体和舒张影像矩阵之间的原始空间关系。图像配准后，根据三维（3D）中质量质心的位移计算了从端向到端面各段的位移量。DIAMOND 显示，与眼道相邻的基底心肌段经历最高的机械变形，并且最易受到多索布辛引起的心脏损伤。总体而言，DIAMOND 为斑马鱼胚胎中除传统喷射分数 （EF） 之外在生理和病理条件下的分段心脏力学提供了新见解。
Introduction
化疗引起的心脏毒性和随后的心力衰竭是化疗中止的主要原因之一。因此，心脏功能评估在心脏毒性的鉴定中起着至关重要的作用，更重要的是，在预测化疗后的早期心脏损伤方面起着至关重要的作用。然而，目前心脏功能评估的方法遇到了限制。方法，如左心室弹出分数（LVEF）只提供全局和经常延迟的心脏力学受伤后3，4。组织多普勒成像提供段状心肌变形信息，但存在显著的观察者内和观察者间变异性，部分原因在于超声波光束角度依赖5。二维（2D）斑点跟踪采用B型超声心动图，理论上消除了角度依赖，但其精度受到平面外运动6的限制。因此，在研究和临床环境中都缺乏量化分段心脏功能的严格方法。
在此背景下，我们开发了一种4D定量方法，用于分析分段心脏功能，将心肌机械变形（DIAMOND）的位移分析命名为”MOND”，以确定3D空间中心肌质量质心的位移载体。我们应用DIAMOND在体内评估心脏功能和多索鲁比辛引起的心脏毒性与斑马鱼（达尼奥雷里奥）作为动物模型，选择由于它们再生心肌和高度保守的发育基因7。我们进一步比较了段段 DIAMOND 位移与全球喷射分数 （EF） 测定和二维应变后多索鲁比素处理。通过结合DIAMOND位移与4D光片荧光显微镜（LSFM）获得渲染胚胎斑马鱼心脏，DIAMOND表明，基底心肌段毗邻眼道发生最高的机械变形，是最容易急性多索布辛心脏损伤8。
Protocol
Representative Results
Discussion
对分段心肌功能进行量化的严格策略对于评估传统EF以外的心脏力学至关重要，众所周知，这是心肌损伤1、4、12的麻木不仁和延迟指标。因此，人们对早期心肌变化的标记越来越感兴趣，越来越多的文献支持心肌变形参数作为预测心室功能障碍4，13的早期指标。左心室（LV）应变的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本工作由美国心脏协会资助16SDG30910007和18CDA34110338，并由国家卫生研究院拨款HL083015、HL111437、HL118650和HL129727。
Materials
| 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 |
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