2.4 Ca2+ imaging of isolated atrial myocytes and HL-1 cells
The Ca2+ imaging of isolated mouse atrial myocytes was performed as previously described (Chen, Xu, Wu, Kranias, et al., 2018; Chen, Xu, Wu, Guo, et al., 2018). Mouse atrial myocytes were isolated from the three groups following a Langendorff-free procedure (Ackers-Johnson et al., 2016). Isolated atrial myocytes were loaded with 5 μM Fluo-4 AM (Invitrogen, Carlsbad, CA, USA) diluted in 20% Pluronic F-127 DMSO at 5 µM final concentration in Tyrode buffer (NaCl 140 mM, KCl 5 mM, HEPES 5 mM, NaH2PO4 2 mM, MgCl2 1 mM, CaCl2 2 mM, glucose 10 mM, pH 7.4) for 10 min. The cells were then washed with Tyrode’s solution with 1.8 mM Ca2+, washed again with fresh Tyrode’s buffer, and maintained in the buffer during confocal Ca2+ imaging. Line-scan imaging (1.82 ms/line) of Ca2+ transients was performed using a Zeiss LSM 800 confocal microscope (Zeiss, Oberkochen, Germany). The imaging was performed by focusing on a single cell. Isoproterenol (1 μM) was added to the cells after the baseline had stabilized. Sparks were evaluated using the spark master plugin in the ImageJ software. F-F0/F0 was also calculated using the ImageJ software. Additionally, 3D images were obtained using ImageJ plugin software, which is an interactive 3D surface plot.
HL-1 cells were seeded in a coated glass bottom dish (24×32 mm, 0.16–0.19 mm, No.1-S; Matsunami) using the same methods as for the HL-1 cell culture. To keep the solution conditions constant, all Ca2+ imaging experiments were carried out by immersing the glass-bottom dish in the medium. The seeded HL-1 cells were then treated with Fluo-4 for 10 min. After washing with fresh medium, the cells were incubated for 10 min in a CO2 incubator. The imaging was performed by focusing on a single cell. The scanning laser line was focused on the single cell and was positioned to include the nucleus. The frequency of abnormal Ca2+ waves was a calculation of how many cells out of all the cells showed the occurrence of abnormal Ca2+ waves.