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

Figures

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  Figure 1.. Morphology of adult cardiomyocytes from mouse, rat, and rabbit in different culturing conditions. (A) Flow chart of workflow for performing FRET assay on adult mouse cardiomyocytes. (B) Bright-field images of adult rabbit cardiomyocytes at 0- and 40-h time points that were maintained in DMEM. (C) Bright-field images of adult rat cardiomyocytes at 0- and 40-h time points that were maintained in M1018 containing 10 mM BDM. (D) Bright-field images of adult mouse cardiomyocytes at 0- and 40-h time points maintained in M1018 alone, M1018 with 10 mM BDM, or M1018 with 25 µM blebbistatin. All the bright-field images were captured at 20× magnification. Scale bar = 100 µm. (E) Sarcomere length measurements of cardiomyocytes at the normal time of infection (0 h) and 40 h later. (F and G) Western blot scans (F) and analysis (G) at the normal time of infection (0 h) and 40 h later of proteins important in proper functionality of cardiomyocytes. PKA RIIα, protein kinase A regulatory subunit IIα; SERCA2A, sarcoplasmic-endoplasmic reticulum calcium ATPase 2A. *, P ≤ 0.05 by unpaired t test. Error bars represent ±SEM.

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  Figure 2.. Media optimization to eliminate autofluorescent properties in adult cardiomyocyte culture. (A) CFP images of adult mouse cardiomyocytes 40 h after infection with the PKA FRET biosensor, AKAR3, maintained in M1018 with different doses of blebbistatin. Images were recorded at 40× magnification. Scale bar = 100 µm. (B) CFP images of adult mouse cardiomyocytes 40 h after infection with AKAR3 maintained in M1018 with 6.25 µM blebbistatin, 0.2% BSA, and 10 mM HEPES as indicated. Images were recorded at 40× magnification. Scale bar = 100 µm. (C) Bright-field images, recorded at 20× magnification at the time of infection (0 h) and 40 h later, of myocytes cultured in M1018 with 10 mM HEPES and 0.2% BSA, and either 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin. Scale bar = 100 µm. (D) Bright-field and CFP images collected at the time of infection (0 h) and 40 h later. Images were recorded at 40× magnification. Scale bar = 100 µm.

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  Figure 3.. Blebbistatin improves morphology of adult mouse cardiomyocytes during extended culture. (A) Bright-field images of adult mouse cardiomyocytes every 10 h when cultured in M1018 + HEPES + BSA with or without 6.25 µM blebbistatin. (B) Bright-field images displaying the morphology of adult mouse cardiomyocytes at previously recommended time points with either 6.25 µM blebbistatin or 500 nM cytochalasin D in the medium. Bright-field images were photographed at 20× magnification. Scale bar = 100 µm. (C) CFP images of adult mouse cardiomyocytes 25 h after infection with AKAR3 when cultured in medium containing either 6.25 µM blebbistatin or 500 nM cytochalasin D. CFP images were collected at 40× magnification. Scale bar = 100 µm. (D and E) Mouse myocytes cultured with blebbistatin and cytochalasin D were costimulated with 10 µM forskolin (FSK) and 100 µM IBMX 25 h after infection with AKAR3 biosensor. The normalized FRET ratios over time are plotted as an average trace, and maximal increases in AKAR3 FRET ratio are depicted in the bar graph. ***, P ≤ 0.001 by unpaired t test. Error bars represent ±SEM.

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  Figure 4.. Time-dependent expression of the PKA FRET biosensor, AKAR3, in adult mouse and rabbit cardiomyocytes. (A and B) Bright-field and corresponding CFP fluorescence images of adult mouse (A) and rabbit (B) cardiomyocytes at different time points of culture as indicated. All images were acquired at 40× magnification. Scale bar = 100 µm.

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  Figure 5.. Optimization of the expression time of AKAR3 for reproducible FRET measurements in adult cardiomyocytes. (A–D) Adult mouse and rabbit cardiomyocytes expressing the PKA biosensor, AKAR3, were costimulated with 10 µM forskolin (FSK) and 100 µM IBMX. The traces depict time courses of changes in FRET ratio, and the maximal increases of FRET ratio are plotted in the bar graphs. *, P ≤ 0.05; ***, P ≤ 0.001 by one-way ANOVA followed by post hoc Bonferroni test. (E) WT mouse myocytes cultured in medium containing either 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin that were expressing the PKA biosensor, AKAR3, were stimulated with 10 µM FSK + 100 µM IBMX, and the average FRET ratios over time were plotted as a trace. (F) The average maximal increases in AKAR3 FRET ratio in myocytes from part E were plotted. Significance was calculated by unpaired t test. Error bars represent ±SEM.

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  Figure 6.. Application of FRET measurements in transgenic adult mouse cardiomyocytes and with a variety of FRET biosensors. (A) WT and β1KO mouse myocytes expressing the PKA biosensor, AKAR3, were stimulated with 100 nM isoproterenol (ISO) followed by 10 µM FSK + 100 µM IBMX, and the average FRET ratios over time were plotted as a trace. (B) The average maximal increases in AKAR3 FRET ratio in WT and β1KO myocytes were plotted. **, P ≤ 0.01 by one-way ANOVA followed by post hoc Bonferroni test. (C) Normal chow (NC) and HFD mouse myocytes expressing the PKA biosensor, AKAR3, were stimulated with 100 nM isoproterenol (ISO) or 10 µM FSK + 100 µM IBMX; the average FRET ratios over time were plotted as a trace. (D) The average maximal increases in AKAR3 FRET ratio in NC and HFD myocytes were plotted. ****, P ≤ 0.0001 by one-way ANOVA followed by post hoc Bonferroni test. (E) Adult mouse cardiomyocytes expressing the cGMP biosensor, cGI500, for 45–46 h were stimulated with 50 µM SNP. FRET ratio is plotted over time. (F) Adult mouse cardiomyocytes expressing the CaMKII biosensor, Camui, for 45–46 h were stimulated with 200 µM Ca2+. FRET ratio is plotted over time. (G) Adult mouse cardiomyocytes…