Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium.

Irina Trautsch,Eriona Heta,Wolfram-Hubertus Zimmermann,Poh Loong Soong,Elif Levent,Dörthe M Katschinski,Manuel Mayr,Ivan Bogeski,Viacheslav O Nikolaev

doi:10.3389/fphys.2019.00272

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.

Highlights

Reactive oxygen species (ROS) can be damaging to cells if produced in excess, and contribute to physiological signaling within and between cells (Sies et al, 2017)
Reactive oxygen species are produced by two major mechanisms: (1) as byproduct of the electron transport chain function in mitochondria (Bertero and Maack, 2018) and (2) via the catalytic activity of NADPH oxidases (NOXs; Zhang et al, 2013)
We first generated TSA cells stably expressing Grx1-roGFP2 and mito-Grx1-roGFP2 by co-transfection of linearized donor vectors encoding for the respective sensors and TALEN-expression vectors to verify compartment-specific sensor location

Summary

Introduction

Reactive oxygen species (ROS) can be damaging to cells if produced in excess, and contribute to physiological signaling within and between cells (Sies et al, 2017). Reactive oxygen species are produced by two major mechanisms: (1) as byproduct of the electron transport chain function in mitochondria (Bertero and Maack, 2018) and (2) via the catalytic activity of NADPH oxidases (NOXs; Zhang et al, 2013). Activity of NOX5 is postulated to contribute to redox signaling in cardiac fibroblasts (Zhang et al, 2013), demonstrating cell type specific ROS production in the heart and suggesting cell type specific ROS signaling. In contrast to classical dyes for ROS quantification - such as dichlorodihydrofluorescein diacetate (H2DCFDA), Amplex R Red or boronate based probes (Rezende et al, 2018) - transgenic sensors allow for stable, long-term measurements of ROS, such as H2O2 (Belousov et al, 2006; Gutscher et al, 2009; Morgan et al, 2016) and the redox potential of ROS scavengers, such as glutathione (Gutscher et al, 2008)

Methods

Results

Conclusion