建立一种结构逼真的心肌细胞的有限元几何模型研究蜂窝结构在心肌系统生物学中的作用
1Cell Structure and Mechanobiology Group,University of Melbourne, 2Systems Biology Laboratory, Melbourne School of Engineering,University of Melbourne, 3Department of Biomedical Engineering,University of Melbourne, 4School of Mathematics and Statistics, Faculty of Science,University of Melbourne, 5Department of Engineering Science,University of Auckland, 6Advanced Microscopy Facility, Bio21 Molecular Science and Biotechnology Institute,University of Melbourne, 7ARC Centre of Excellence in Convergent Bio-Nano Science and Technology,University of Melbourne, 8School of Medicine, Faculty of Medicine, Dentistry and Health Sciences,University of Melbourne, 9Living Systems Institute,University of Exeter
Summary
该协议概述了一种新的方法, 以建立一个空间详细的有限元模型的细胞内体系结构的电子显微镜和共焦显微图像。通过对钙信号和生物能学的案例研究, 证明了这个空间细节模型的威力。
Abstract
随着三维 (3D) 成像技术的出现, 如电子层析成像、串行块面扫描电子显微镜和共焦显微术, 科学界在亚微米上对大型数据集有前所未有的访问权。在健康和疾病中伴随心肌细胞功能变化的结构重塑的分辨率。然而, 这些数据集已被用于研究细胞结构重塑在心肌功能中的作用。本协议的目的是概述如何使用高分辨电子显微镜和共焦显微图像, 建立一个精确的心肌细胞有限元模型。一个详细和准确的细胞结构模型有很大的潜力, 提供新的洞察力的心肌生物学, 比单独的实验可以获得。这种方法的威力在于它能够从两种不同的心肌细胞超微结构成像模式中融合信息, 从而形成一个统一的、详细的心肌细胞模型。本协议概述了将成人雄性大鼠心肌细胞的电子层析成像和共焦显微图像整合的步骤 (名称为特定品种的白化大鼠), 以建立一个半小节有限元心肌模型。该过程生成一个3D 的有限元模型, 其中包含一个精确的, 高分辨率的描述 (35 nm 的顺序) 的分布线粒体, 肌原纤维和兰尼碱受体簇, 释放必要的钙的心肌细胞收缩从肌网状网络 (SR) 入肌原纤维和胞浆室。此处生成的模型不包含横向小管结构或肌网状网络的细节, 因此是心肌细胞的最小模型。然而, 该模型已经被应用于基于模拟的研究中的细胞结构在钙信号和线粒体生物能学的作用, 这是说明和讨论使用两个个案研究, 提出以下详细的协议。
Introduction
心脏的励磁收缩耦合 (ECC) 是指心肌细胞膜的电刺激与细胞在每次心跳过程中随后的机械收缩之间的重要而复杂的耦合。数学模型在发展对相互关联的生物化学过程的定量理解方面发挥了关键作用, 这些进程调节了动作电位1、胞浆钙信号2、生物能学3 以及随后的收缩力的产生。当其中一个或几个生化过程发生改变时, 这些模型也成功地预测了心跳的变化4,5。心肌细胞的高度组织超微结构越来越被认为对正常收缩功能和整体心脏起着关键作用。事实上, 心脏超微结构成分的形态学和组织的变化与疾病条件的生物化学变化 (如肥厚6、心力衰竭7和糖尿病心肌病8) 平行发生。无论这些结构变化是轻微的, 适应性, 或病理反应变化的生物化学条件仍然很大程度上未知的9。生物学中形式和功能之间固有的紧密耦合意味着实验研究不能比结构重塑和心肌细胞功能之间的相关性提供更深的洞察力。新一代的数学模型可以将子细胞成分的结构组装, 连同经过研究的生物化学过程结合起来, 对关系进行全面、定量的理解是必要的。在心肌细胞的结构、生物化学和收缩力之间。该协议描述了可用于生成结构精确的心肌细胞有限元模型的方法, 可用于此类调查。
过去十年在3D 电子显微术10、共焦11和超分辨率显微镜12上取得了重大进展, 为纳米尺度和微尺度组装提供了前所未有的高分辨率洞察力。心肌细胞的分胞成分。最近, 这些数据集被用来生成心肌细胞超微结构的计算模型13,14,15,16。这些模型使用了一个已建立良好的工程模拟方法, 称为有限元方法17, 以创建有限元计算网格的生物化学过程和心肌收缩可以模拟。但是, 这些模型受显微镜方法可以在图像数据集中提供的分辨率和细节的限制。例如, 电子显微学可以生成纳米级的细胞结构细节, 但很难确定图像内的特定蛋白质, 这将是必要的创建模型。另一方面, 超分辨率光学显微术可以提供高对比度图像在分辨率上的 50 nm 的顺序只有一个选择少数分子成分的细胞。只有通过将互补信息与这些成像方式相结合, 才能切实地探索功能对结构变化的敏感性。相关光和电子显微术仍然不是一个常规的程序, 它仍然会受到限制, 只有有限数量的成分可能被染色在免疫荧光的观点, 并与电子显微镜的看法相关联。
此协议提供了一种新的方法18 , 使用统计方法19分析和计算保险丝光显微学信息关于离子通道的空间分布的电子显微镜信息在其他心脏超微结构成分, 如肌原纤维和线粒体。这产生了一个有限元模型, 可用于生物化学过程的生物物理模型, 以研究心肌细胞分胞组织对调节心肌细胞收缩的生物化学过程的作用。例如, 该协议可用于建立健康和链脲佐菌素诱导的糖尿病心肌细胞模型, 研究结构重塑对糖尿病动物模型中观察到的心肌细胞功能的影响8。该方法还说明了该方法的统计性质的另外一个优点: 该法能产生多实例的有限元几何, 它可以密切模仿实验观察到的细胞结构变化。
作为一项概述, 协议步骤包括: (一) 制备用于电子显微术的心脏组织, 生成具有足够分辨率和对比度的3D 图像;(二) 使用3D 电子显微重建和图像分析软件 (称为 IMOD20) 重建和分割3D 图像栈, 从电子显微镜数据中提取;(iii) 使用 iso2mesh21生成使用分段数据作为输入的有限元网格;(iv) 利用新的算法和编码将离子通道的分布映射到有限元网格上。
在议定书内概述了每一步骤的方法的前提, 并在所附数字中提供了代表性的结果。概述了如何使用生成的空间详细模型来研究在 ECC 中钙的空间动力学, 以及线粒体生物能学。讨论了该议定书目前的一些局限性, 以及正在进行的新的发展, 以克服这些限制, 并进一步促进对细胞结构在心脏系统生物学中的作用的定量理解。还讨论了如何将这些方法推广到其他细胞类型的有限元模型中。
如果用户能够访问预先存在的电子显微图像栈, 则此协议的使用者可能跳过步骤1和步骤2的重建部分。有意与更有经验的电子 microscopists 合作获取其数据的用户可能希望与专家讨论和比较步骤1中的固定和染色程序, 以确定最佳的获取协议。
Protocol
这里所描述的所有方法都得到了奥克兰大学动物伦理委员会和加利福尼亚大学圣地亚哥机构动物护理和使用委员会的批准, 组织协议最初是在那里制定的。 1. 实验准备 根据表 1, 在 pH 值为7.4 的情况下, 准备0.15 米和 0.3 m 钠常使用缓冲器的库存解决方案。 使用表7.4 中列出的组件, 将戊二醛固定剂 (2% 多聚甲醛 (粉煤灰) + 2.5% 戊二醛 + 0.2% 单宁酸?…
Representative Results
图 2通过图 7提供了本协议中几个关键步骤的代表性结果: (一) 可视化和重新定向组织块以进行横断面电子显微观察;(二) 生成3D 电子显微图像栈;(iii) 将亚细胞超微结构分割为感兴趣的器;(iv) 利用 iso2mesh 生成有限元网格;(v) 模拟 RyR 簇在网格上的真实分布;(vi) 接着将这一空间分布映射为计算网格上的空间密度。 <p class="jove_content…
Discussion
上述协议概述了生成一种新的心肌细胞超微结构有限元几何模型的关键步骤。该方法使不同显微学 (或原则上, 其他数据) 模式的计算融合, 形成了一个更全面的心肌细胞动力学计算模型, 其中包括空间单元结构的细节。目前还没有其他的协议可以创建这样一个心肌细胞模型。
步骤1概述了一个灌注固定的协议。或者, 心脏可以切除, 解剖成小块, 浸泡在固定体。然而, 这一过程必?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到新西兰皇家学会快速入门补助金 11-UOA-184, 人类前沿科学项目研究赠款 RGP0027/2013 和澳大利亚研究委员会发现项目赠款 DP170101358。
Materials
| Materials | |||
| Sodium chloride | Sigma-Aldrich | 746398 | |
| Calcium chloride | Sigma-Aldrich | C8106 | |
| Magnesium chloride | Sigma-Aldrich | M2393 | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | |
| Potassium chloride | Sigma-Aldrich | P5405 | |
| Dextrose | Sigma-Aldrich | D9434 | |
| Sodium hydroxide | Sigma-Aldrich | S8045 | |
| Probenecid | Sigma-Aldrich | P8761 | |
| 2,3-Butanedione monoxime | Sigma-Aldrich | B0753 | |
| 25% Glutaraldehyde EM Grade (500 ml bottle) | Merck | 354400-500ML | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| Tannic Acid | Sigma-Aldrich | 403040-500G | 100g EM grade |
| Sodium cacodylate | Sigma-Aldrich | C0250 | |
| Phosphate-buffered saline (PBS) | Sigma-Aldrich | P4593 | |
| Osmium Tetroxide | Sigma-Aldrich | 75632-10ML | 4% in water, 5 ml bottle (or 10 ml bottle also available) |
| Uranyl Acetate | EM Sciences | 22400 | 25g bottle |
| Potassium Ferrocyanide | Merck Millipore | 104973 | |
| Toluene blue | Sigma-Aldrich | T3260 | |
| Borax | Sigma-Aldrich | S9640 | also termed sodium borate |
| Ethanol | Sigma-Aldrich | 792780 | Diluted to different percentages with pure water |
| Acetone | EM Sciences | RT10017 | |
| Resin kit | EM Sciences | 14040 | ACM Durcupan works well |
| Hydrochloric acid | Sigma-Aldrich | H9892 | 1Normal solution |
| Equipment | |||
| Ultramicrotome | Leica | EM UC7 | |
| Transmission electron microscope | ThermoFisher Scientific | Tecnai F30 | http://www.leica-microsystems.com/ |
| Retort stand | Proscitech | T752 | |
| Tubing | BioStrategy | 75831-346 | for langendorff perfusion apparatus, 3 mm diameter is recommended but not essential |
| Stopcocks | SDR | QP13813 | for langendorff tubing; product is only an example, user can select any |
| retort stand clamps | Proscitech | T715 | |
| Plastic syringes | SDR | QPC1108 | for solutions on langendorff apparatus |
| Cannulation silk suture, 7-0 | TeleFlex | 15B051000 | for tieing heart on langedorff apparatus |
| Cannula | Made from 3 mm outer-diameter steel needle | ||
| Rubber petri dish mat | Proscitech | H068 | for use as cutting board during fixed-heart dissection |
| Razor blades | Proscitech | L056 | for cutting fixed-heart into small blocks for EM processing |
| Glass bottles | BioStrategy | 89000-236 | for storing solutions during tissue fixation and processing for EM |
| Beakers | BioStrategy | 213-0477 | for storing solutions temporarily and during perfusion |
| Scintillation vials | BioStrategy | 548-2170 | for tissue samples during EM processing |
| Dissection kit | Proscitech | T161 | for animal dissection |
| Syringe Filters | Proscitech | WS3-02225S | for purification of Uranyl Acetate |
| Aluminium/silver foil baking cups | From any baking products store | ||
| Dupont Diamond knife | BioStrategy | 102680-780 | 35 degree angle version produces best sections. |
| Colloidal Gold | BBI Solutions | EM. GC15 | 15 nm colloidal gold |
| EM mesh grids | Proscitech | GCU150 | a variety of sizes can be tested: GCU150h, GCU200h for example |
| Plastic disposal pippettes | Proscitech | LCH20 | best to use plastic disposables especially when working with resin |
| Software | |||
| SerialEM | University of Boulder | tomography acquisition | |
| MATLAB | MathWorks | https://www.mathworks.com/products/matlab.html | |
| IMOD | University of Boulder | image alignment and segmentation | |
| iso2mesh | available at http://iso2mesh.sourceforge.net | ||
| Fiji or similar image processing software | ImageJ | Fiji is Just Image J | available at https://fiji.sc for manipulation of binary image stacks |
| RyR-Simulator codes/data | CellSMB group | available at https://github.com/CellSMB/RyR-simulator | |
| CardiacCellMeshGenerator | CellSMB group | comes with RyR-Simulator under folder "gui-version" | |
| R-statistics software | R-project | Download from https://www.r-project.org | |
| spatstat | R-project | install via R program | |
| rgl | R-project | install via R program | |
| doparallel | R-project | install via R program | |
| foreach | R-project | install via R program | |
| doSNOW | R-project | install via R program | |
| iterators | R-project | install via R program |
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Cite This Article
Rajagopal, V., Bass, G., Ghosh, S., Hunt, H., Walker, C., Hanssen, E., Crampin, E., Soeller, C. Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology. J. Vis. Exp. (134), e56817, doi:10.3791/56817 (2018).