Development of a genetically encoded model for the sensing of glutathione redox potential in human embryonic stem cell-derived cardiomyocytes and fibroblasts

Abstract

Redox alterations due to increased ROS production in heart cells have been implicated in several cardiovascular diseases, such as ischemia, hypertrophy, and myocardial infarction, but may also play a crucial role for the wanted and unwanted effects of pharmacological stimulation. Genetically encoded biosensors allow for the visualization of redox changes at real-time in a quantitative manner, but have not yet been applied to human heart models. In this study, the hypothesis was tested that the cytosolic glutathione (GSH) redox sensor Grx1-roGFP2 can be applied to record the GSH redox state in human cardiomyocytes and fibroblasts. The cytosolic glutathione redox sensor Grx1-roGFP2 was stably introduced into human embryonic stem cell-derived cardiomyocytes (HES2-CM) and human foreskin fibroblasts (HFF) via lentiviral transduction. The kinetics of the Grx1-roGFP2 sensor were determined in a cell type specific manner in HES2-CM and HFF under increasing concentrations of H2O2 (0.1 - 1,000 µmol/L), diamide (1 - 1,000 µmol/L), and DTT (0.1 - 1 mmol/L). This identified a more reduced EGSH in HES2-CMs (-289 ± 1 mV; n=56) compared to HFFs (-269 ± 2 mV; n=18). Subsequently, GSH redox state alterations were investigated upon ROS stimulation with angiotensin II and pharmacological stimulation with cardioactive reference compounds (levosimendan, omecamtiv mecarbil). To investigate cell specific GSH redox changes in a tissue context, engineered heart muscles (EHM) were generated by mixing either (i) Grx1-roGFP2-HFFs and naive HES2-CMs or (ii) Grx1-roGFP2 HES2-CMs and naive HFFs in a collagen hydrogel. Force generating EHM were formed within 20 days and were subjected to an acute oxidative (H2O2) and reductive (DTT) challenge. GSH oxidation and reduction could be recorded optically and associated with a reduced and increased contractile performance in EHM. Taken together, GSH redox state can be recorded optogenetically in living human cardiomyocytes and fibroblasts in classical monolayer and EHM culture. Differences in EGSH suggest difference in redox related signalling in cardiomyocytes and fibroblasts which may have to be considered when interpreting redox responses to drugs or other stimuli at the whole heart level.