Deciphering cellular signals in adult mouse sinoatrial node cells

Figures

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  Figure 1. Design and validation of the adult mouse SAN cell culturing method (A) Steps for obtaining and culturing adult mouse SAN cells. Some of the images were created with Biorender.com. (B–D) Brightfield images of adult mouse SAN cells in M1018 media alone or containing 10 mM 2,3-Butanedione 2-monoxime (BDM), 6.5 μM blebbistatin (Bleb), or 20 μM (S)-nitro blebbistatin (nitro Bleb) at 0 and 40 h (hr) in culture. Scale bar, 10 mm. Scatterplots of cell length (C; n = >48 cells from three preparations per condition) and % spindle-shaped cells (D; n = ≥5 images from three preparations per condition) in the different media conditions at 0 and 40 h in culture. Statistical differences were assessed with two-tailed Kruskal-Wallis with Dunn's test for comparison between all groups and two-tailed Mann-Whitney test for comparisons between the 0 and 40 h groups. Exact p values are available in Table S1. (E) Representative spontaneous action potential (AP) traces and scatterplot of firing rate, maximum diastolic potential (MDP), and peak amplitude obtained from adult mouse SAN cells freshly dissociated or cultured in M1018 media +20 μM (S)-nitro blebbistatin for 40 h (n = 10 cells per condition). AP…

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  Figure 2. cAMP signals in freshly isolated and cultured CAMPERCM SAN cells (A) Cartoon showing the strategy for the generation of the CAMPERCM mouse. (B) Diagram of the cAMP biosensor expressed in the CAMPERCM mouse in the cAMP-free and cAMP-bound form. (C) Representative population of freshly isolated and cultured adult SAN cells from the CAMPERCM mouse displaying YFP and CFP fluorescence (similar results were observed in two independent preparations). Scale bar, 10 mm. (D) Exemplary pseudo-colored FRET ratio images of freshly isolated and cultured CAMPERCM SAN cells before and after 10 μM forskolin (fsk). Scale bar, 10 μm. (E) Average FRET ratio traces (mean, solid lines; SEM, shadow) before and after application of 10 μM fsk in freshly isolated (fresh) and cultured (cult) CAMPERCM SAN cells. (F) Average FRET ratio traces (mean, solid lines; SEM, shadow) before and after application of 100 nM isoproterenol (iso), 1 μM histamine (hist), 1 nM CGRP, and 1 μM adenosine (ade). (G) Scatterplot summarizing freshly dissociated and cultured CAMPERCM SAN cells ΔR/R0 FRET responses to fsk (n = >9 cells from two preparations per condition; P = 0.295 with two-tailed Mann-Whitney test for comparison…

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  Figure 3. Expression of FRET-based sensors in adult mouse SAN cells (A) Diagram of the cytosolic FRET-based CUTie biosensor (Surdo et al., 2017). (B–D) Representative brightfield image of a wild-type adult mouse SAN cell. The cell was not infected with the CUTie biosensor as confirmed by the lack of YFP and CFP associated fluorescence in the accompanied images, as well as the lack of changes in YFP and CFP fluorescence intensity in response to 10 μM forskolin (fsk; C, D; n = 5 cells from two preparations). Scale bar, 10 μm. (E) Representative images of a wild-type adult mouse SAN cell showing robust YFP and CFP-associated fluorescence and pseudo-colored YFP/CFP ratio images before and after application of 10 μM fsk. Scale bar, 10 μm. (F) Time profile of YFP and CFP fluorescence, and YFP/CFP ratio changes in response to 10 μM fsk. (G) Scatterplot quantifying changes in YFP and CFP fluorescence, and YFP/CFP ratio after application of forskolin (n = 10 cells from three preparations). Data represent mean ± SEM.

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  Figure 4. 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.…

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  Figure 5. Detection of different signaling networks in adult mouse SAN cells (A) Schematic diagram of cGMP reporter cG1500. (B) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 μM ANP or 100 μM SNAP and scatterplots of ΔR/R0 treatment effect (n = >6 cells from three preparations per condition). A, ANP; S, SNAP. (C) Schematic diagram of the PKA activity reporter AKAR3. (D) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol or 10 μM forskolin and scatterplots of ΔR/R0 treatment effect (n = >6 cells from three preparations per condition). I, isoproterenol; F, forskolin. (E) Schematic diagram of the CaMKII activity reporter Camui. (F) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 1 μM PE or 200 μM calcium and scatterplots of ΔR/R0 treatment effect (n = >6 cells from three preparations per condition). P, PE; C, calcium. (G) Schematic diagram of the PKD activity reporter DKAR. (F) Average FRET ratio traces (mean, solid lines; SEM = shadow) in response to 10 μM histamine or 500 nM PDBu and scatterplots of ΔR/R0 treatment effect (n = >6 cells from three preparations per condition). Statistical…

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  Figure 6. cAMP and cGMP signaling in adult mouse SAN cells during HF (A) Representative whole heart images taken at 8 weeks after sham and TAC (HF) surgery. Scatterplot of (B) heart weight to body weight (HW/BW) ratio (P = 0.0004 with unpaired t test) and (C) lung weight to body weight (LW/BW) ratio (P = 0.0214 with unpaired t test) from sham and HF mice (n = 5 hearts per condition). (D) Representative images of sham and HF hearts at the parasternal short axis. (E) Summary scatterplot data of corrected left ventricular (LV) mass in sham and HF mice (P = 0.0005 with unpaired t test; n = 5 hearts per condition). (F) Representative M-mode echocardiographic images for sham and HF mice. Scatterplot data from echocardiography for (G) fractional shortening (P = 0.0009 with unpaired t test), (H) ejection fraction (P = 0.0029 with unpaired t test), and cardiac dimensions (I) from sham and HF mice (n = 5 hearts per condition). LVAW;d, left ventricular arterial wall—diastole (P = 0.0029 with t test); LVAW;s, left ventricular arterial wall—systole (P = 0.8560 with t test); LVID;d, left ventricular interior diameter—diastole (P = 0.0007 with t test); LVID;s, left ventricular interior diameter—systole…