All reagents and compounds were purchased from either Fujifilm Wako Pure Chemical (Osaka, Japan), Thermo Fisher Scientific (Waltham, MA, USA), or Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise.
The human iPSC-derived “iCell Cardiomyocytes2” (iPSC-CMs) used in this study and their culture method are the same as those reported in our previous study . Briefly, iPSC-CMs were purchased from Fujifilm Cellular Dynamics International (Tokyo, Japan). The purity of cardiomyocytes was ≥99%. The cells were seeded onto collagen I-coated 96-well plates (Corning, Inc., Corning, NY, USA) at a density of 93,750 cells/cm2 using iCell Cardiomyocytes Plating Medium, according to the manufacturer’s protocol. The cells were grown and maintained in 5% CO2 at 37 °C. Four hours after seeding, the plating medium was replaced with the manufacturer-provided iCell Cardiomyocytes Maintenance Medium. The maintenance medium was refreshed every two days throughout the experiment.
Compound preparation and stimulation
After the iPSC-CMs were cultured for 5–6 d, they were stimulated with sunitinib with or without XMU-MP-1 for 72 h. Both sunitinib and XMU-MP-1 were dissolved in 100% (v/v) dimethyl sulfoxide (DMSO). The compounds were added to the wells of a 96-well plate to obtain the final concentrations 0.3 μM, 1, μM, or 3 μM for either sunitinib or XMU-MP-1. The final DMSO concentration was 0.1% (v/v). The cells were then cultured with each compound for 72 h. To evaluate the effect of drug washout on sarcomere structure, the sunitinib-containing medium was removed and replaced with fresh drug-free medium after 72 h of sunitinib stimulation.
Paraformaldehyde (PFA) fixation and immunostaining
The PFA fixation and immunostaining processes were performed according to our previously reported methods . After drug stimulation and washout, cells were fixed with 2% PFA for 15 min at 37 °C then washed twice with PBS. After blocking the cells with blocking buffer [1× PBS Tween-20 (28,352; Thermo Fisher Scientific) containing 1.5% (v/v) bovine serum albumin (A9205; Sigma-Aldrich)] for 30 min at room temperature (22–25 °C), the cells were incubated with a polyclonal antibody against rabbit alpha-actinin (AB90776; Abcam, Cambridge, UK), diluted with blocking buffer to a final concentration of 2 μg/mL, at 4 °C for 18 h. To analyze YAP1 nuclear localization, we used a rabbit monoclonal antibody against active YAP1 (AB205270; Abcam), diluted with blocking buffer to a final concentration of 0.2 μg/mL, at 4 °C for 18 h. After discarding the primary antibody solution, cells were washed twice with PBS and incubated with a secondary antibody solution, including goat anti-rabbit IgG (H + L) secondary antibody (A11034, Alexa Fluor 488, 1:400) and Hoechst 33342 (1:800; Thermo Fisher) at 4 °C for 1 h. Finally, cells were washed twice with PBS, and each well was filled with PBS before performing high-content analysis (HCA).
Assessment of cardiac sarcomere structure and YAP1 nuclear localization using HCA
The protocol for the analysis of the cardiac sarcomere structure has been described previously . Briefly, automated fixed-cell fluorescent images of immunostained cells were obtained using the IN Cell Analyzer 6500 device (GE Healthcare Japan, Tokyo, Japan) using a 60× or 4× objective lens. To analyze the sarcomere structure, nine image fields per well were acquired using the 60× objective lens. To analyze YAP1 nuclear localization, six image fields per well were acquired using the 4× objective lens. For all image analyses, we used the IN Cell Developer Tool Box software (GE Healthcare). The number of cell nuclei per image field was calculated by observing the pattern of Hoechst-stained nuclei, and the total number of cell nuclei per well were determined. For quantification of the sarcomere morphology, we measured the lengths of all the Z lines of the sarcomeres observed in each cell by analyzing the image exhibiting the immunofluorescence signal of alpha-actinin, then we calculated the average length of all recognized Z lines per cell. Next, to determine whether the observed cells were healthy or morphologically damaged, based on the average length of all recognized Z lines per cell, we counted the number of cells per field that differed concomitantly according to changes in the cutoff value, to determine the average length of Z lines of cells. By searching for an optimal cutoff value, we defined cells with an average Z line sarcomere length > 3.1 μm as cells with a “ well-organized sarcomere.” Then, we counted the number of cells with well-organized sarcomeres per well using the analysis software, and determined the ratio of the number of cells with well-organized sarcomeres to the total number of cells observed in each well, which we defined as the “Healthy sarcomere index (HSI)” in all experiments. To determine the HSI, approximately 200 cells were analyzed in each well (because of high magnification (× 60)), one well was used for each data point, and five or six wells were used as the data for one group (n = 5–6 wells/group) Similarly, the ratio of HSI in the treatment group to that in the control group was calculated. For quantification of the YAP1 nuclear localization, immunofluorescence intensity of YAP1 in the cytoplasm and nuclei, respectively, were measured. Then, cells having a significantly more intense signal of YAP1 in the nuclei than in the cytoplasm were defined as cells showing nuclear localization of YAP1. Then, the degree of nuclear localization of YAP1 was determined by calculating the ratio of the number of cells showing nuclear localization of YAP1 to the total number of cells observed in each well and expressed as the relative ratio to the control group. For the quantitation of nuclear localization of YAP1, approximately 4000 cells were analyzed in each well (because of low magnification (× 4)), one well was used for each data point, and five or six wells were used as the data for one group (n = 5–6 wells/group). Since the iPSC-CMs from Fujifilm Cellular Dynamics International have been previously reported to be predominantly mononuclear cells , we conducted image analyses in all the experiments assuming that the iPSC-CMs are mononuclear cells.
All results are presented as mean ± SD values and all graphs were created using the GraphPad Prism 8 software. Statistical analyses were performed using EXSUS2014 software. The HSI and degree of YAP1 nuclear localization between the DMSO and the drug-treated group were compared using Williams’ test or Shirley-Williams’ test, depending on whether the variances were heterogeneous or not. Statistical significance was set at p < 0.025. Student’s t-test or the Wilcoxon rank sum test was conducted to perform comparisons between DMSO and 3 μM sunitinib-treated groups in Fig. 3D; p < 0.05 was considered statistically significant.