For imaging, cells were seeded in glass-bottom eight-well µ-slides (Ibidi). CMs were plated on eight-well glass-bottom µ-slides (Ibidi) coated with a 1:30 dilution of growth factor reduced Matrigel (Corning) in serum-free MC plating medium. CMs were given time to settle and attach on µ-slides for at least 2 h at 37 °C and 5% CO2. Then, MC plating medium was changed to imaging buffer (pH 7.4, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [Hepes], 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.5% bovine serum albumin [BSA]) Transfection of HEK293AD was done using Effectene (Qiagen) according to the manufacturer’s instructions and imaged 2 d after transfection.

Prior to imaging, transfected HEK293ADs, CMs, CM-hiPSCs, and H9c2 cells were preincubated for 40 min to 1 h with either 100 nM CGP 20712 (to image β2-AR) or 50 nM ICI 118,551 (to image β1-AR) diluted in the appropriate imaging buffer. Then, 5 or 50 nM JE1319 as indicated was diluted in imaging buffer and was directly added to the cells (with CGP 20712 or ICI 118,551 as described) for 40 min to 1 h. During the last 15 min of ligand incubation, 0.25× CellMask Green Plasma Membrane Stain (Invitrogen) was added to the cells in case of H9c2 cells and CM-hiPSCs. Untransfected HEK293ADs and A431 cells did not undergo the antagonist preincubation step.

After all incubations, cells were washed three times (except CMs, which were washed only once) using imaging buffer and also imaged in this buffer. CMs were imaged in imaging buffer containing the respective β-AR antagonist as well as 50 µM para-aminoblebbistatin (Optopharma) to inhibit spontaneous CM contractions. Finally, cells were imaged on an SP8 confocal laser scanning microscope (Leica) under physiological conditions (37 °C, 5% CO2, 85% humidity) using a sample incubator (Stage Top Chamber; OKOlab).

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