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. 2020 Jan 14;10:209. doi: 10.1038/s41598-019-56597-3

Figure 5.

Figure 5

AMPK is a positive regulator of sarcomere-cytoskeleton filament interactions. (A–C) Immunoprecipitation of MYH7 with TnT is shown, using cell lysates from DCM iPSC-CMs, healthy controls and TnT KO. Interaction of TnT with MYH7 was assessed by immunoprecipitation using a TnT-specific antibody. (A) Representative membrane scans for immunoprecipitation of TnT and MYH7 are shown. (B) Comparable amounts of TnT-WT and TnT- R173W were immunoprecipitated from lysates of healthy control and DCM iPSC-CMs. (C) Binding of MYH7 to DCM-TnT-R173W is reduced, compared to healthy controls. Bargraphs show signal from bound IP fractions following normalization for input in each group for input and loading control. Shown are averages of 2 experiments for control (WT, n = 3 cell lines), DCM (TnT-R173W, n = 3 cell lines) and TnT KO (n = 1 cell line); input and loading control are shown in Supplementary Fig. 7A. *P < 0.05, **P < 0.01 (one sample t- and Wilcoxon test). Data are expressed as mean ± sem. (D–E) AMPK activity in DCM versus healthy control iPSC-CMs measured via a phospho-AMPKα (Thr172)-specific antibody. Following treatment with A-769662, AMPK activity in DCM iPSC-CMs is significantly increased, compared to control vehicle (DMSO). (D) Representative membrane scans are shown. (E) Quantification of (D). Shown are averages of 4 experiments for control (WT, n = 3 cell lines), DCM (TnT-R173W, n = 3 cell lines) and TnT KO (n = 1 cell line); **P < 0.01 and ns, not significant (one-way ANOVA and Sidak’s multiple comparison test) data are expressed as mean ± sem. (F–I) Co-localization of AMPK with MYH7 and vimentin is reduced in DCM iPSC-CMs compared to healthy controls and is recovered following AMPK activation via A- 769662. Immunostaining and confocal images (scale bar, 20 μm) as well as corresponding ImageJ-based quantifications are shown. Line scans show striation patterns for sarcomeric distribution of TnT (y-axis, fluorescence intensity in arbitrary units; x-axis, distance in μm). (F) Co-localization of AMPK with MYH7 and (G) Quantification of (F), ***P < 0.001 for WT control vehicle vs. DCM control vehicle, **P < 0.01 for DCM control vehicle vs. DCM-A-769662 and WT control vehicle vs. DCM-A-769662, P = not significant (one-way ANOVA and Sidak multiple comparison test). Analysis was performed for WT healthy control, n = 2 cell lines and n = 68 cells; DCM, n = 1 cell line and n = 49 cells; and DCM A-769662, n = 2 cell lines and n = 74 cells. (H) Co-localization of AMPK with vimentin. Scale bar, 20 μm. (I) Quantification of (H), ***P < 0.001 for DCM control vehicle versus DCM-A-769662 (one-way ANOVA and Sidak multiple comparison test) WT healthy control, n = 2 cell lines and n = 38 cells; DCM, n = 1 cell line and n = 23 cells; and DCM A-769662, n = 1 cell line and n = 14 cells. Data are shown as mean ± sem.