Fig 1.
Characterization of wild-type and BRAF iPSC-hECTs.
(A) Sorting paradigm for construction of defined iPSC-hECTs; (B) Photograph of wild-type (left) and mutant (right) tissues after 6 days in culture; (C) longitudinal section of wild-type (left) and mutant (right) tissues stained with hematoxylin and eosin after 12 days in culture; (D) Post deflections of both tissue types on day 6 at 2 Hz pacing frequency; (E) Cross-sectional area (mean±SD) of wild-type (open bars, n = 7) and mutant (filled bars, n = 4) tissues; (F) Molecular analysis of mutant and wild-type tissues performed on day 12 (n = 3 for each tissue type).
Fig 2.
Twitch force characteristics of wild-type and BRAF-mutant hECTs.
(A) Cartoon of iPSC-derived hECT force data illustrating twitch characteristic parameters. (B-G) Mean (±SD) twitch parameters for wild-type (open bars, n = 7) and BRAF mutant (solid bars, n = 5) iPSC-hECTs tested on culture days 6 and 11, including developed force (B), 50% twitch duration (C), time to 50% contraction (D), time to 50% relaxation (E) and the maximum rates of contraction (F) and relaxation (G). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3.
Electrical properties of of wild-type and BRAF-mutant hECTs.
(A) Developed force versus frequency relationship of iPSC-derived hECTs at culture day 6 (left) and day 11 (right); (B) maximum pacing frequency of wild-type and mutant tissues; (C) spontaneous beating frequency; (D) The minimum voltage necessary for pacing for both tissues; (E) Relative chronotropic response to isoproterenol (normalized to baseline beating frequency) with fitted nonlinear regression model. (F) Twitch rate variability plots of each tissue type with a paced control for comparison. Each color represents a different tissue. (G) Quantification of twitch rate variability by the mean distance of each point on the twitch rate variability plots from each cluster centroid. Error bars represent standard error (A) or standard deviation (B-G) Open symbols and bars are wild-type hECTS (n = 7), filled symbols and bars are BRAF-mutant hECTs (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4.
Investigating longitudinal changes in hECT functional phenotype.
(A) Principal component analysis and k-means clustering of tissues paced at 2 Hz on day 6 (left) and day 11 (right). (B) Developed force of the wild-type tissues increased significantly over day 6 and day 11, while mutant tissues did not. The lack of a significant change from day 6 to day 11 appeared to be due to a greater increase in diastolic force of mutants from day 6 to 11 than wild-type tissues, while systolic force increased approximately by the same amount for both tissue types. (C) Passive Young’s modulus determined by uniaxial stretch measurements on days 12–15 (n = 4 per tissue type). * p < 0.05 between tissues types, † p < 0.05 between day 6 and day 11 for wild-type tissues.
Table 1.
Summary of component loadings on the principal components that explain 95% of the variance on experimental day 6.
Table 2.
Summary of component loadings on the principal components that explain 95% of the variance on experimental day 11.