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Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium

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Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium

Irina Trautsch et al. Front Physiol.

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.

Keywords: GSH; cardiomyocytes; engineered human myocardium; fibroblasts; optogenetics; redox-reporters; roGFP; stem cells.

Figures

Figure 1
Figure 1
TSA cells express Grx1-roGFP2 in a compartment specific manner. (A) Live cell imaging of roGFP-reporter (GFP), TMRM (mitochondria) and Hoechst (DNA) in TSA cells demonstrated the anticipated cytosolic and mitochondrial localization of Grx1-roGFP2 and mito-Grx1-roGFP2, respectively; note that the GFP signal in the Grx1-roGFP2 condition fills the whole cell, whereas the mito-Grx1-roGFP2 signal superimposes with the spatially confined perinuclear TMRM signal and presents as a distinct rim around the nucleus because of the high nucleus to cytosol ratio in TSA cells. Scale bars: 50 μm. (B) Representative Western blot analysis to confirm enrichment of the respective roGFP2 reporters in cytosolic (c) and mitochondria (m) compartments. The mitochondrial compartment is characterized by proteins of the electron transport chain complexes I - IV (SDHB: Succinate dehydrogenase complex iron sulfur subunit B, MTCO1: Cytochrome c oxidase I, UQCRC2: ubiquinol-cytochrome c reductase core protein II, ATP5A: ATP synthase subunit alpha).
Figure 2
Figure 2
TALEN-modified HES2 express functional Grx1-roGFP2. (A) Flow cytometry analysis of GFP positive cells in genetically naïve (HES2 wt) and TALEN-modified Grx1-roGFP2 and mito-Grx1-roGFP2 HES2. (B) Representative Western blot analysis to confirm enrichment of the respective roGFP2 reporters in cytosolic (c) and mitochondria (m) compartments. The mitochondrial compartment is characterized by proteins of the electron transport chain complexes I - IV (NDUFB8: NADH: ubiquinone oxidoreductase subunit B8, SDHB: Succinate dehydrogenase complex iron sulfur subunit B, MTCO1: Cytochrome c oxidase I, UQCRC2: ubiquinol-cytochrome c reductase core protein II, ATP5A: ATP synthase subunit alpha). (C) Oxidation and reduction of roGFP2 results in an inversely correlated shift in fluorescence intensity under 408 and 488 nm excitation. (D) Fluorescence intensity shift in exemplary false colored images of HES2 mito-Grx1-roGFP2 cells at t = 0 s and t = 150 s upon oxidative (H2O2) and reductive (DTT) stimulation. Red: 400 nm excitation, green: 500 nm excitation, scale bars: 100 μm. (E–H) Change of roGFP2 fluorescence signal in HES2 Grx1-roGFP2 (E,G) and HES2 mito-Grx1-roGFP2 (F,H) as a function of time under oxidative (H2O2, E, F) and reductive (DTT, G, H) stimulation. H2O2 or DTT were added at 30 s. Grx1-roGFP2: n = 241 cells (300 μmol/L H2O2), n = 205 cells (100 μmol/L H2O2), n = 203 cells (10 μmol/L H2O2), n = 52 cells (1 μmol/L H2O2), n = 197 cells (10 mmol/L DTT), n = 264 cells (3 mmol/L DTT), n = 189 cells (1 mmol/L DTT), n = 58 cells (0.1 mmol/L DTT), n = 53 cells (0.01 mmol/L DTT); mito-Grx1-roGFP2: n = 182 cells (300 μmol/L H2O2), n = 301 cells (100 μmol/L H2O2), n = 305 cells (10 μmol/L H2O2), n = 57 cells (1 μmol/L H2O2), n = 193 cells (10 mmol/L DTT), n = 252 cells (3 mmol/L DTT), n = 274 cells (1 mmol/L DTT), n = 56 cells (0.1 mmol/L DTT), n = 57 cells (0.01 mmol/L DTT).
Figure 3
Figure 3
HES-derived cardiomyocytes express functional roGFP2 sensors, but show an impaired sarcomere phenotype. Change of roGFP2 fluorescence signal in hCM from HES2 Grx1-roGFP2 (A) and HES2 mito-Grx1-roGFP2 (B) as a function of time under oxidative (H2O2) and reductive (DTT) stimulation. Grx1-roGFP2: n = 176 cells (100 μmol/L H2O2), n = 170 cells (1 mmol/L DTT), mito-Grx1-roGFP2: n = 325 cells (100 μmol/L H2O2), n = 325 cells (1 mmol/L DTT). (C) Immunofluorescence analysis after staining for α-actinin and DNA (Hoechst); GFP: roGFP2 reporter signal. Scale bars: 50 μm. Insets: magnifications of α-actinin labeled sarcomere structures, scale bars: 20 μm.
Figure 4
Figure 4
Lentiviral expression of Grx1-roGFP2 reports redox changes in differentiated cardiomyocytes and fibroblasts. (A) Brightfield (left) and GFP fluorescence (right) images after lentiviral transduction of hCM and hFF; scale bars: 200 μm. (B) Flow cytometry analysis of transduction efficiency in hCM (47 ± 7%, n = 7) and hFF (72 ± 6%, n = 8); p < 0.05, unpaired, two-tailed Student’s t-test. Change of roGFP2 fluorescence signal in hCM (C) and hFF (D) as a function of time under oxidation by H2O2 at indicated concentrations (n = 46–71 [hCM]; n = 19–43 [hFF]). Change of roGFP2 fluorescence signal in hCM (E) and hFF (F) as a function of time under reduction by DTT at indicated concentrations (n = 46–85 [hCM]; n = 21–37 [hFF]).
Figure 5
Figure 5
Cell type specific imaging of Grx1-roGFP2 in EHM. (A) EHM were constructed from defined mixtures of genetically naïve and Grx1-roGFP2 expressing hCM or hFF in a collagen hydrogel to create tissue for cell type specific redox potential imaging. (B) Photograph and fluorescence image of EHM expressing Grx1-roGFP2 (GFP). Analysis of force of contraction (FOC) under isometric conditions and electrical field stimulation (1.5 Hz); maximal inotropic capacity was evaluated under increasing extracellular calcium concentrations: (C) EHM composed of Grx1-roGFP2 and genetically naïve (wt: wild type) hCM with genetically naïve hFF (n = 17/33); (D) EHM composed of Grx1-roGFP2 and genetically naïve (wt: wild type) hFF with genetically naïve hCM (n = 36/42). Change of roGFP2 fluorescence signal in EHM with Grx1-roGFP2 expressing hCM (E) and hFF (F) as a function of time under oxidation by H2O2 (1 mmol/L) and reduction by DTT (1 mmol/L).

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