Figure 2

Characterizations of the Myom2-RFP ESC line as a cardiomyocyte maturation reporter. (a) A schematic protocol for cardiac differentiation. (b) Myom2-RFP expression during a prolonged culture. Data is presented as means ± SD (n = 4). One-way ANOVA with post-hoc Tukey HSD test; § P < 0.01, † P < 0.0001. Fluorescence intensity is presented as arbitrary unit (a.u.). (c) Representative fluorescence images of RFP⁻ and RFP⁺ cardiomyocytes at day 28. A yellow line indicates the line scan region displayed in (d). Myom2-RFP (red); α-actinin (green); Nuclei (blue). Scale bar, 20 μm. (d) Corresponding line scan for the altered pattern of Myom2-RFP and α-actinin in a sarcomere. (e) Comparisons of structure and morphology between RFP⁻ and RFP⁺ cardiomyocytes (n > 65). Sarcomere length, cell area, perimeter length, cell length, cell width, and aspect ratio were examined. For violin plots, black lines in the white boxes show the medians, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, and polygons represent density estimates of data and extend to extreme values. Student’s t test; *P < 0.05, ^§P < 0.01, ^#P < 0.001, ^†P < 0.0001. (f) Proportion of mononuclear and binuclear cells in RFP⁺ and RFP⁻ PSC-CMs at day 21 and 28 (n > 65). Chi-square Test; ^#P < 0.001, ^†P < 0.0001. (g–i) Calcium transients of RFP⁻ and RFP⁺ cardiomyocytes: (g) Representative time-lapse images of calcium transients for RFP⁻ and RFP⁺ cardiomyocytes (scale bar, 20 μm), and (h) corresponding spatially averaged profiles of cytosolic calcium transients evoked by electrical field stimulation at 1 Hz, at day 28. (i) Mean values of time to peak, peak amplitude, and decay time of Ca²⁺ transients (n > 30). Student’s t test; ^#P < 0.001, ^†P < 0.0001. (j–m) Sarcomere shortening assays for PSC-CMs using Myom2-RFP: (j) Representative images for sarcomere shortening with measuring regions as indicated by yellow bars and (k) corresponding line scans of sarcomere lengths. (l) Sarcomere shortening profile of RFP⁺ cardiomyocytes. (m) Normalized sarcomere lengths and percent of sarcomere shortening of RFP⁺ cardiomyocytes (n = 38).