Wagner Eva, Brandenburg Sören, Kohl Tobias, Lehnart Stephan E
Heart Research Center Goettingen; Clinic of Cardiology & Pulmonology, University Medical Center Goettingen; German Center for Cardiovascular Research (DZHK) partner site Goettingen.
Heart Research Center Goettingen; Clinic of Cardiology & Pulmonology, University Medical Center Goettingen.
J Vis Exp. 2014 Oct 15(92):e51823. doi: 10.3791/51823.
In cardiac myocytes a complex network of membrane tubules--the transverse-axial tubule system (TATS)--controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca(2+) release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
在心肌细胞中，一个由膜小管组成的复杂网络——横-轴小管系统（TATS）——控制着深入的细胞内信号传导功能。虽然外表面膜和相关的TATS膜成分似乎是连续的，但脂质和蛋白质含量存在显著差异。在心室肌细胞（VMs）中，某些TATS成分高度丰富，有助于形成直线状的小管网络和规则分支的三维结构。据认为，外周TATS成分将动作电位从细胞表面传播到数千个遥远的细胞内肌浆网（SER）膜接触域，从而激活细胞内钙（2+）释放单元（CRUs）。与VMs不同，TATS膜在心房肌细胞（AMs）中的组织和功能作用有显著差异，且了解较少。综上所述，对健康和患病心肌细胞中TATS膜网络进行定量结构表征是更好地理解功能可塑性和病理生理重组的必要前提。在这里，我们提出了一套策略组合方案，用于对活的VMs和AMs中的TATS膜网络进行直接定量分析。为此，我们在小鼠VMs和/或AMs的原代细胞分离过程中，加入关键的质量控制步骤以及用于TATS膜荧光成像的直接膜染色方案。使用优化的共聚焦或超分辨率TATS图像处理工作流程，生成二值化和骨架化数据，用于定量分析TATS网络及其成分。与先前发表的间接区域聚集图像分析策略不同，我们的方案能够直接表征特定成分，并利用高通量和开放获取的软件工具得出活心肌细胞中TATS膜网络的复杂生理特性。总之，该组合方案策略可轻松应用于生理心肌细胞适应或疾病变化期间的TATS网络定量研究、不同心脏或骨骼肌细胞类型的比较、转基因模型的表型分析以及药理或治疗干预。
