Fig 1: Discrete cAMP pools in adult mouse SAN cells(A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 µM forskolin (fsk) + 100 µM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 µM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ?R/R0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (*) was considered at P < 0.05. Exact p values are available in Table S3. Data represent mean ± SEM.