Multiparametric imaging of spatio-temporal cAMP signaling, transmembrane potential, and intracellular calcium in the intact heart

Figures

• 

  Figure 1. Quadruple-parametric FRET and dual optical mapping system (A) Schematic of the quadruple-parametric imaging system illustrating the optics used and the light path for cAMP FRET, Vm, and Ca2+ signals. (Bi.), (Biii.) Excitation and (Bii.), (Biv.) emission spectra of the three parameters—cAMP FRET (CFP and YFP), Rhod-2 or X-Rhod-1 (Ca2+), and RH237 (Vm) fluorescence, illustrating the spectral separation of the three signals as implemented in this system. Dotted lines: excitation wavelength, solid lines: emission wavelengths, long-dotted vertical lines: dichroic mirrors, shaded boxes: filters.

• 

  Figure 2. Validation of FRET imaging (A) Representative images of a cardiac-specific CAMPER mouse heart showing the raw intensity of CFP and YFP fluorescence upon CFP excitation. (B) Cross-sectional intensity of CFP and YFP from RA to apical left ventricle (yellow dashed arrow from A). (C) FRET maps (R, CFP/YFP) at baseline (R0) and during acute 1.5 μM NE bolus infusion. (D) Cross-sectional intensity of baseline FRET (R0) from RA to ventricle (yellow dashed arrow from A). (E) ΔFRET (R/R0) maps at baseline and during acute 1.5 μM NE bolus infusion. (F) Mean scatterplots from RV base (blue) and LV apex (orange) of R0. Paired t test, ∗p < 0.05, N = 4 hearts.

• 

  Figure 3. Effects of blebbistatin on cAMP FRET signals (A) Representative temporal CFP and YFP fluorescence. (B) ΔFRET response in a CAMPER mouse heart following application of a 1.5 μM bolus NE, recorded first without and then with 10 μM blebbistatin. (C) Average ΔFRET responses between hearts. (D) Max R/R0 and (E) 50% time to decay in response to bolus of 1.5 μM NE recorded first without and then with 10 μM blebbistatin. Paired t test, ∗p < 0.05, N = 4 hearts.

• 

  Figure 4. Validation of spectral separation (Ai.) Imaging of hearts stained with Rhod-2 or (Ci) X-Rhod-1 only and (Aii.) (Cii) following with RH237. (B) Representative FRET acquisition of example hearts stained with no dyes, Rhod-2 or (D) X-Rhod-1 only and with RH237 during an acute bolus of 0.5 μM NE infusion. Kolmogorov-Smirnov test, No dye vs. Rhod-2, p = 0.39; No dye vs. Both, p = 0.39; Rhod-2 vs. Both, p = 0.95. No dye vs. X-Rhod-1, p > 0.99; No dye vs. Both, p > 0.99; X-Rhod-1 vs. Both, p > 0.99, N = 3 hearts (Rhod-2); N = 1 heart (X-Rhod-1).

• 

  Figure 5. Demonstration of whole-heart cAMP and Vm-Ca2+i response following β-adrenergic stimulation (A) Example maps of action potential duration (APD80). (B) Calcium transient duration (CaTD50) and (C) ΔFRET images during acute 0.5 mM NE infusion (scale bars, 3 mm). (D) Temporal whole-heart CFP and YFP fluorescence responses in CAMPER mouse hearts after application of a 0.5 μM bolus NE. (E) Temporal whole-heart ΔFRET changes with heart rate and (F) whole-heart APD80, CaTD50 changes in response to 0.5 μM NE. Paired t test, ∗p < 0.05 vs. time = 0 s, N = 3 hearts. The same example heart, including APD80, CaTD50, and ΔFRET images, is shown in Figure 6 to demonstrate regional parameter quantification.

• 

  Figure 6. Regional effects of b-adrenergic stimulation on cAMP responsiveness in the CAMPER mouse heart (A) Representative ΔFRET ratio images from Figure 5C showing the spatiotemporal kinetics of cAMP activity (scale bars, 3 mm) and (Bi.) example FRET ratio trace and (Bii.) mean scatterplots at time = 40 s (black arrow) in response to bolus of 0.5 μM NE from different regions of the heart (blue = RV base; orange = apex). (C) Representative CaTD50 (from Figure 5B) and (E) APD80 (from Figure 5A) maps from the heart in (A) (top) with change in CaTD50 and APD80 vs. baseline (ΔCaTD50 and ΔAPD80) (bottom). (Di.) Example CaTD50 and (Fi.) APD80 percentage change trace over time and (Dii. and Fii.) mean scatterplots from RV base (blue) and apex (orange) following bolus of 0.5 μM NE. Paired t test, ∗p < 0.05, N = 4 hearts.

• 

  Figure 7. Atrial cAMP and Vm-Ca2+i response following β-adrenergic stimulation (A) Example atrial preparation for optical mapping in a Langendorff-perfused CAMPER heart. Left atrium = LA, right atrium = RA. Scale bars, 3 mm. (B) Representative Vm and Ca2+ optical traces in the atria before and after a bolus of 1.5 μM NE. Black arrow indicates slight ventricular signal bleed through due to light scattering. (C) Representative ΔFRET map and (D) Vm activation map in the atria after an acute bolus of 1.5 μM NE. Scale bars, 3 mm. (E) Averaged (mean = solid lines; SEM = shadow) temporal ΔFRET ratio traces in the sinoatrial node (SAN) with heart rate during bolus of 1.5 μM NE. N = 3 hearts.

• 

  Figure 8. Effects of sympathetic and parasympathetic activation on whole-heart cAMP activity (A) Representative ΔFRET images showing the spatiotemporal kinetics of cAMP activity (scale bars = 3 mm) in response to bolus of 0.5 μM NE. (B) 20 μM tyramine and (C) 1.5 μM acetylcholine and averaged (mean = solid line; SEM = shadow) temporal ΔFRET traces from different regions of the heart (green = LA; blue = RV base; orange = apex). N = 3–4 hearts.