Abstract
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)-nitroblebbistatin to a minimal essential medium containing 10 mM HEP ES 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1567-1582 |
| Number of pages | 16 |
| Journal | Journal of General Physiology |
| Volume | 150 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 1 2018 |
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ASJC Scopus subject areas
- Physiology
Cite this
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 Kevin.
In: Journal of General Physiology, Vol. 150, No. 11, 01.11.2018, p. 1567-1582.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes
AU - Reddy, Gopireddy Raghavender
AU - West, Toni M.
AU - Jian, Zhong
AU - Jaradeh, Mark
AU - Shi, Qian
AU - Wang, Ying
AU - Chen-Izu, Ye
AU - Xiang, Yang Kevin
PY - 2018/11/1
Y1 - 2018/11/1
N2 - 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)-nitroblebbistatin to a minimal essential medium containing 10 mM HEP ES 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.
AB - 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)-nitroblebbistatin to a minimal essential medium containing 10 mM HEP ES 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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UR - http://www.scopus.com/inward/citedby.url?scp=85056266101&partnerID=8YFLogxK
U2 - 10.1085/jgp.201812119
DO - 10.1085/jgp.201812119
M3 - Article
C2 - 30242036
AN - SCOPUS:85056266101
VL - 150
SP - 1567
EP - 1582
JO - Journal of General Physiology
JF - Journal of General Physiology
SN - 0022-1295
IS - 11
ER -