Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium
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 o...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2019-04-01
|
| Series: | Frontiers in Physiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fphys.2019.00272/full |
| _version_ | 1828338915478601728 |
|---|---|
| author | Irina Trautsch Irina Trautsch Eriona Heta Eriona Heta Poh Loong Soong Poh Loong Soong Elif Levent Elif Levent Viacheslav O. Nikolaev Viacheslav O. Nikolaev Ivan Bogeski Ivan Bogeski Dörthe M. Katschinski Dörthe M. Katschinski Manuel Mayr Wolfram-Hubertus Zimmermann Wolfram-Hubertus Zimmermann |
| author_facet | Irina Trautsch Irina Trautsch Eriona Heta Eriona Heta Poh Loong Soong Poh Loong Soong Elif Levent Elif Levent Viacheslav O. Nikolaev Viacheslav O. Nikolaev Ivan Bogeski Ivan Bogeski Dörthe M. Katschinski Dörthe M. Katschinski Manuel Mayr Wolfram-Hubertus Zimmermann Wolfram-Hubertus Zimmermann |
| author_sort | Irina Trautsch |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T22:34:41Z |
| format | Article |
| id | doaj.art-cddc89d2ea8c468497753b9d0edd52e5 |
| institution | Directory Open Access Journal |
| issn | 1664-042X |
| language | English |
| last_indexed | 2024-04-13T22:34:41Z |
| publishDate | 2019-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Physiology |
| spelling | doaj.art-cddc89d2ea8c468497753b9d0edd52e52022-12-22T02:26:48ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2019-04-011010.3389/fphys.2019.00272435174Optogenetic Monitoring of the Glutathione Redox State in Engineered Human MyocardiumIrina Trautsch0Irina Trautsch1Eriona Heta2Eriona Heta3Poh Loong Soong4Poh Loong Soong5Elif Levent6Elif Levent7Viacheslav O. Nikolaev8Viacheslav O. Nikolaev9Ivan Bogeski10Ivan Bogeski11Dörthe M. Katschinski12Dörthe M. Katschinski13Manuel Mayr14Wolfram-Hubertus Zimmermann15Wolfram-Hubertus Zimmermann16Institute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyInstitute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyInstitute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyInstitute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyInstitute for Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Hamburg, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyInstitute for Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyInstitute for Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, GermanyKing’s British Heart Foundation Centre, King’s College London, London, United KingdomInstitute of Pharmacology & Toxicology, University Medical Center Göttingen, Göttingen, GermanyDZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, GermanyRedox 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.https://www.frontiersin.org/article/10.3389/fphys.2019.00272/fulloptogeneticsengineered human myocardiumredox-reportersstem cellscardiomyocytesfibroblasts |
| spellingShingle | Irina Trautsch Irina Trautsch Eriona Heta Eriona Heta Poh Loong Soong Poh Loong Soong Elif Levent Elif Levent Viacheslav O. Nikolaev Viacheslav O. Nikolaev Ivan Bogeski Ivan Bogeski Dörthe M. Katschinski Dörthe M. Katschinski Manuel Mayr Wolfram-Hubertus Zimmermann Wolfram-Hubertus Zimmermann Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium Frontiers in Physiology optogenetics engineered human myocardium redox-reporters stem cells cardiomyocytes fibroblasts |
| title | Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium |
| title_full | Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium |
| title_fullStr | Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium |
| title_full_unstemmed | Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium |
| title_short | Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium |
| title_sort | optogenetic monitoring of the glutathione redox state in engineered human myocardium |
| topic | optogenetics engineered human myocardium redox-reporters stem cells cardiomyocytes fibroblasts |
| url | https://www.frontiersin.org/article/10.3389/fphys.2019.00272/full |
| work_keys_str_mv | AT irinatrautsch optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT irinatrautsch optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT erionaheta optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT erionaheta optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT pohloongsoong optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT pohloongsoong optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT eliflevent optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT eliflevent optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT viacheslavonikolaev optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT viacheslavonikolaev optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT ivanbogeski optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT ivanbogeski optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT dorthemkatschinski optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT dorthemkatschinski optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT manuelmayr optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT wolframhubertuszimmermann optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium AT wolframhubertuszimmermann optogeneticmonitoringoftheglutathioneredoxstateinengineeredhumanmyocardium |