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Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger sp...

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Autores principales: Reddy, Gopireddy Raghavender, West, Toni M., Jian, Zhong, Jaradeh, Mark, Shi, Qian, Wang, Ying, Chen-Izu, Ye, Xiang, Yang K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219686/
https://www.ncbi.nlm.nih.gov/pubmed/30242036
http://dx.doi.org/10.1085/jgp.201812119
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author Reddy, Gopireddy Raghavender
West, Toni M.
Jian, Zhong
Jaradeh, Mark
Shi, Qian
Wang, Ying
Chen-Izu, Ye
Xiang, Yang K.
author_facet Reddy, Gopireddy Raghavender
West, Toni M.
Jian, Zhong
Jaradeh, Mark
Shi, Qian
Wang, Ying
Chen-Izu, Ye
Xiang, Yang K.
author_sort Reddy, Gopireddy Raghavender
collection PubMed
description FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10–15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes.
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spelling pubmed-62196862019-05-05 Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes Reddy, Gopireddy Raghavender West, Toni M. Jian, Zhong Jaradeh, Mark Shi, Qian Wang, Ying Chen-Izu, Ye Xiang, Yang K. J Gen Physiol Research Articles FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10–15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes. Rockefeller University Press 2018-11-05 /pmc/articles/PMC6219686/ /pubmed/30242036 http://dx.doi.org/10.1085/jgp.201812119 Text en © 2018 Reddy et al. http://www.rupress.org/terms/https://creativecommons.org/licenses/by-nc-sa/4.0/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Research Articles
Reddy, Gopireddy Raghavender
West, Toni M.
Jian, Zhong
Jaradeh, Mark
Shi, Qian
Wang, Ying
Chen-Izu, Ye
Xiang, Yang K.
Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
title Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
title_full Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
title_fullStr Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
title_full_unstemmed Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
title_short Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
title_sort illuminating cell signaling with genetically encoded fret biosensors in adult mouse cardiomyocytes
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219686/
https://www.ncbi.nlm.nih.gov/pubmed/30242036
http://dx.doi.org/10.1085/jgp.201812119
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