HiExM shows altered nuclear morphology of cardiomyocytes treated with low doses of doxorubicin
a. hiPSC-CMs imaged post-expansion in HiExM. b. Example images of hiPSC-CM nuclei after dox treatment for 24 hrs before (top) and after (bottom) expansion. c. Example images of hiPSC-CM nuclei taken before (top) and after (bottom) expansion. d. Heatmaps of nuclei in c showing pixel groupings for subsequent analysis. Heatmaps were generated from the image mask after dilation, such that the outer edge represents a contour just beyond the nuclear periphery. e. Relative Hoechst intensity plotted as a function of pixel position relative to the edge of the dilated mask in pre-(top) and post-(bottom) expansion nuclei. Colored curves represent dox concentrations or DMSO (ctrl). Shaded regions represent SEM for n = 56, 71, 64, 92, and 62 nuclei in the pre-expansion images and n = 4 replicates in the expanded case (118, 111, 110, 113, and 77 nuclei analyzed for DMSO control, 1 nM, 10 nM, 100 nM, and 1 µM Dox, respectively). f. Insets of plots in e highlighting the nuclear periphery. g. Rate-of-change analysis of curves for pre-(left) and post-(right) expansion images. Outer edge values were obtained by determining the maximum values of the derivatives for each condition, and inner edge values were obtained by determining the minimum values of the derivatives within the domain 0 - 0.4. h. Curvature analysis for pre-(left) and post-(right) expansion data. Values represent the minima of the second derivative for curves within the domain 0 – 0.4. i. Average Hoechst intensities plotted as gradients for each dox concentration. Scale bar represents 10 percent distance from the edge of the nucleus to the center. Error bars represent SEM as in e. * denotes significance from an independent two-sample t-test (p < 0.05). *Scale bar represents biological scale assuming 4x expansion.