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

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)-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.

A Gs-coupled purinergic receptor boosts Ca2+ influx and vascular contractility during diabetic hyperglycemia

“…Intriguingly, cAMP synthesis in response to elevated glucose and the P2Y 11 agonist NF546 was significantly larger in human myocytes compared to mouse cells (glucose: 0.077 ± 0.003 in human vs 0.045 ± 0.002 in mouse; NF546: 0.083 ± 0.005 in human vs 0.045 ± 0.004 in mouse; p<0.05). The reasons for this are currently unclear but may include intrinsic differences associated with species (human vs mouse), blood vessels (human adipose arteries vs mouse aortae) and expression patterns of the biosensor in human vs mouse cells (Reddy et al, 2018), as well as variations in protein expression and receptor/enzyme activity (e.g. P2Y 11 , AC and PDE expression/activity) in human vs mouse arterial myocytes.…”

Insulin receptor substrates differentially exacerbate insulin-mediated left ventricular remodeling

“…Thirty-six hours after transduction, cells were stimulated with 1 × 10 -5 μM ANP plus or minus the PDE3 inhibitor Cilostamide, PDE5 inhibitor Sildenafil, or PDE9a inhibitor PF-04447943 at various concentrations (1 × 10 -7 M, 1 × 10 -6 M, and 1 × 10 -5 M, respectively). Changes in FRET were measured using live cell microscopy as previously described (5,50).…”

May the Force Not Be With You During Culture: Eliminating Mechano-Associated Feedback During Culture Preserves Cultured Atrial and Pacemaker Cell Functions

“…Most importantly, the use of blebbistatin improved the efficiency of adenovirus-mediated gene transfer in the cultured myocytes compared to BDM (Kabaeva et al, 2008). Based on this study, use of blebbistatin to eliminate mechano-feedback has significant beneficial effects on the long-term usability of adult myocytes in primary culture, as it reduces ATP production (Kovács et al, 2004;Watanabe et al, 2010) and maintains a stable phosphorylation state in the cultured cardiomyocytes (Reddy et al, 2018;Segal et al, 2019) while avoiding the undesirable effects of BDM on adult myocytes.…”