Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium

Abstract

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.

Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells

“…Moreover, the biosensor Grx1-roGFP2 may be targeted toward different subcellular compartments, such as cytosol [ 36 ] and mitochondria [ 37 ]. Several approaches have been undertaken to express the biosensor Grx1-roGFP2 in cell lines [ 36 ] and tissues [ 38 ], and even transgenic organisms expressing Grx1-roGFP2 have been created [ 39 ]. As far as we know, to date, there is not any publication in the literature that refers to any model of skeletal muscle where the biosensor Grx1-roGFP2 would have been used to determine GSH/GSSG.…”

Extracellular Vesicles in Cardiac Regeneration: Potential Applications for Tissues-on-a-Chip

“…Commonly used genetically encoded sensors include the engineered calcium sensitive GCaMP protein [83] and its many derivatives and the ArcLight voltage sensor [84]. Recent studies have also incorporated sensors to measure glutathione redox state as a proxy for oxidative stress [85].…”

A cell-type and compartment specific analysis of glutathione and hydrogen peroxide

“…Trautsch, I., Heta, E., Soong, P. L., Levent, E., Nikolaev, V. O., Bogeski, I., Katschinski, D. M., Mayr, M., & Zimmermann, W.-H. (2019). Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium.…”

“…In conclusion, the integration and expression of fluorescent redox sensor did not influence pluripotency of the analyzed HES2 lines. (Trautsch et al, 2019).…”

“…Since random integration did not seem to attenuate GFP expression in these cell lines and picked clones were uniformly positive for redox sensor expression, we continued to analyze the sensor function. (Trautsch et al, 2019).…”