Trautsch Irina, Heta Eriona, Soong Poh Loong, Levent Elif, Nikolaev Viacheslav O, Bogeski Ivan, Katschinski Dörthe M, Mayr Manuel, Zimmermann Wolfram-Hubertus
Institute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, Germany.
DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany.
Front Physiol. 2019 Apr 4;10:272. doi: 10.3389/fphys.2019.00272. eCollection 2019.
Redox signaling affects all aspects of cardiac function and homeostasis. With the development of genetically encoded fluorescent redox sensors, novel tools for the optogenetic investigation of redox signaling have emerged. Here, we sought to develop a human heart muscle model for in-tissue imaging of redox alterations. For this, we made use of (1) the genetically-encoded Grx1-roGFP2 sensor, which reports changes in cellular glutathione redox status (GSH/GSSG), (2) human embryonic stem cells (HES2), and (3) the engineered heart muscle (EHM) technology. We first generated HES2 lines expressing Grx1-roGFP2 in cytosol or mitochondria compartments by TALEN-guided genomic integration. Grx1-roGFP2 sensor localization and function was verified by fluorescence imaging. Grx1-roGFP2 HES2 were then subjected to directed differentiation to obtain high purity cardiomyocyte populations. Despite being able to report glutathione redox potential from cytosol and mitochondria, we observed dysfunctional sarcomerogenesis in Grx1-roGFP2 expressing cardiomyocytes. Conversely, lentiviral transduction of Grx1-roGFP2 in already differentiated HES2-cardiomyocytes and human foreskin fibroblast was possible, without compromising cell function as determined in EHM from defined Grx1-roGFP2-expressing cardiomyocyte and fibroblast populations. Finally, cell-type specific GSH/GSSG imaging was demonstrated in EHM. Collectively, our observations suggests a crucial role for redox signaling in cardiomyocyte differentiation and provide a solution as to how this apparent limitation can be overcome to enable cell-type specific GSH/GSSG imaging in a human heart muscle context.
氧化还原信号传导影响心脏功能和内环境稳定的各个方面。随着基因编码荧光氧化还原传感器的发展，用于氧化还原信号传导光遗传学研究的新工具应运而生。在此，我们试图开发一种用于氧化还原变化组织内成像的人类心肌模型。为此，我们利用了：（1）基因编码的Grx1-roGFP2传感器，其可报告细胞内谷胱甘肽氧化还原状态（GSH/GSSG）的变化；（2）人类胚胎干细胞（HES2）；以及（3）工程化心肌（EHM）技术。我们首先通过TALEN引导的基因组整合，生成了在细胞质或线粒体区室中表达Grx1-roGFP2的HES2细胞系。通过荧光成像验证了Grx1-roGFP2传感器的定位和功能。然后，使表达Grx1-roGFP2的HES2细胞进行定向分化，以获得高纯度的心肌细胞群体。尽管能够报告细胞质和线粒体中的谷胱甘肽氧化还原电位，但我们在表达Grx1-roGFP2的心肌细胞中观察到了肌节形成功能障碍。相反，在已经分化的HES2心肌细胞和人包皮成纤维细胞中进行Grx1-roGFP2的慢病毒转导是可行的，且不会损害细胞功能，这在由表达Grx1-roGFP2的特定心肌细胞和成纤维细胞群体构建的EHM中得到了证实。最后，在EHM中证明了细胞类型特异性的GSH/GSSG成像。总的来说，我们的观察结果表明氧化还原信号传导在心肌细胞分化中起着关键作用，并提供了一种解决方案，以克服这一明显的限制，从而在人类心肌环境中实现细胞类型特异性的GSH/GSSG成像。
