Sympathetic Neurons Regulate Cardiomyocyte Maturation in Culture

Figures

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  FIGURE 1. Co-culture protocol. (A) Diagram of cell sources and co-culture protocol. SNs were dissociated from E13.5 mouse sympathetic ganglia. GFP expressing hiPSC-CM were cultured alone (CM-only) or with SN (CM + SN) for 30 days. In other experiments, we included EPI and EC (CM + SN + EPI + EC). EPI were harvested from E13.5 mouse hearts and formed vascular smooth muscle and fibroblasts. ECs were HUVECS. (B–E) Co-cultures immunostained at day 30. (B) GFP-expressing hiSPC-derived CMs (green) were positive for cardiac troponin-T (red). (C) SNs expressed tyrosine hydroxylase (blue). (D) EPI cells showed α-smooth muscle actin (vascular smooth muscle, red) and Col1a1 (fibroblasts, cyan). (E) ECs are marked with von Willebrand factor (white). Scale bars 250 μm.

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  FIGURE 2. Relative gene expression. (A) Example FACS graphs of sorted GFP + CMs after dissociation of co-cultures. (B) Heatmap display of the log fold-change in CM specific genes, normalized to the CM-only culture. Genes are grouped based on their function in CM maturation. n ≥ 2 biological samples per gene.

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  FIGURE 3. CM sarcomere organization and Cx43 expression. (A–C) Sarcomeres stained with α-actinin (red). GFP-expressing hiPSC-CM are shown in green. (A′–C′) Higher magnification of boxed areas in (A–C). (D–E) Sarcomere organization (D) and spacing (E) from analysis of immunofluorescence images; **p <0.01, ***p <0.001, ****p <0.0001. Data are mean ± SD. Welch’s ANOVA with Dunnett’s T3 multiple comparisons test is shown. n = 9 areas from 3 CM-only cultures, n = 16 areas from 5 CM + SN, and n = 14 areas from 4 CM + SN + EPI + EC. (F–H) Immunofluorescence of Cx43 puncta (red) within hiPSC-CM (green). (F′–H′) Higher magnification of boxed regions in (F–H). The Cx43 channel is shown in monochrome to visualize differences among the groups. (I) Total Cx43 area as a fraction of CM area; *p <0.05. Data are mean ± SD. Welch’s ANOVA with Dunnett’s T3 multiple comparisons test is shown. n ≥ 9 areas from five cultures per group. Scale bars 50 μm (A–C) and (F–H), 25 μm (A′–C′) and (F′–H′).

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  FIGURE 4. Ca2+ transients in CMs. (A,B) Sample images of GCaMP6s-expressing hiPSC-CMs in CM-only (A) and CM + SN (B) culture conditions. The white box regions show example ROIs for Ca2+ transient analysis. (C) Example trace of normalized Ca2+ fluorescence (F/F0) in CM-only culture during intrinsic beating. (D) Ca2+ trace of a CM + SN co-culture. (E,F) Amplitude (E) and RT90 (F) of the Ca2+ transients in CM-only and CM + SN; **p <0.01, ****p < 0.0001. Data are mean ± SD. Unpaired t-test is shown. (G,H) Example Ca2+ traces during caffeine injection in CM-only (G) and CM + SN (H) co-cultures. Three example plots are shown for both conditions to demonstrate the range of caffeine responses. Plots are scaled so that the baseline amplitudes appear equal. Arrows indicate the timing of caffeine infusion. (I) The caffeine-induced increase in Ca2+ transient amplitude was quantified relative to the baseline Ca2+ amplitude; ****p < 0.0001. Box plots extend between the 25th and 75th percentile, the median value is shown by a horizontal line, and the whiskers show the entire range. Welch’s t-test is shown. (J) Standard deviation (SD) of local caffeine increases observed across all ROIs from single…