62 Chapter 3 camera (frame rate: 75 fps, shutter speed: 1/150 sec) and motion velocity analysis was performed using Sony Cell Motion Imaging System (SI8000) (Hayakawa et al., 2014). Briefly, motion velocity analysis calculates contraction/relaxation velocity and deformation distance by computationally measuring movement of pixels in the consecutive video images. Since cells are patterned into known surface area (1800 μm2) and attached to hydrogel substrate of known stiffness (10 kPa), maximum contractile force was calculated using Young’s modulus equation. After recordings, cells were fixed immuno-stained with antibodies to cTnT and α-actinin, counterstained with DAPI, and imaged using Zeiss LSM710 confocal microscope. Quantification of sarcomere alignment was performed using FIJI software with an additional analysis package (Orientation J) to determine the angle of z-disc-registered sarcomeric α-actinin. For example, 90 ° angle of z-disc-registered sarcomeric α-actinin represents myofibril alignment along cellular long axis. Biophysical effect examination using hydrogel substrate. To modulate substrate stiffness, polyacrylamide hydrogels were prepared as previously described (Lee et al., 2019, 2020). Briefly, 40% of acrylamide and 2% bisacrylamide stock solution was diluted with deionized water to make polyacrylamide hydrogel precursor solution with the final concentration of acrylamide at 8% and the final concentration of bisacrylamide at 0.06%, 0.15%, and 0.6%, to achieve hydrogel stiffness of 1, 10, and 60 kPa, respectively. The hydrogel precursor solution was sandwiched between two coverslips and photo-crosslinked under light (365 nm, 4 mW/ cm2) for 5 min. After photocrosslinking the hydrogel, one coverslip was peeled off to reveal a hydrogel substrate. Day 24 hiPSC-CMs were plated on the hydrogel substrate and cultured for 5 days in expansion media (B27+CHIR 2μM) for 5 days before fixation. Immunostaining was done as described above. Rabbit-derived Yap antibody was diluted at 1:50. YAP inhibition study. To pharmacologically inhibit YAP activity, a well-known inhibitor, verteporfin (SML0534, Sigma) was administered to day 22 hiPSC-CMs at different concentrations (0.1, 1, 10, 50, 100 μM) for 24 hours. 50, 100 μM verteporfin treatment led to cell death. Next day, the cells were fixed and stained for YAP, TnT, and Ki67 as described above.
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