Fig 2: HCM-affected family and molecular characterization of human tissue and hiPSC-derived CMs from 2D and 3D models APedigree of HCM-affected family carrying a novel ACTN2 mutation (c.740C>T; T247M). Individuals who were genotyped are marked with an apostrophe, and HCM patients are indicated by filled symbols and carriers of this ACTN2 mutation with a (+) sign. Encircled in red with the arrow is the index patient (II.4) from whom the HCM hiPSC line was created.BRepresentative Sanger sequencing results of the ACTN2 locus in the Ctrl, HCM, and HCMrep hiPSCs (higher magnification of the locus shown on the right, red rectangle). Depicted is the mutation on position g.54,208 (c.740C>T), the repair template from g.54,148 to 54,270 (-strand), the according silent mutations encoded on the repair template on position g.54,200 (G>C; -strand) and g.54,245 (C>G; -strand), and sgRNAs A and B.C, DWestern blot analysis of hiPSC-CMs (C) and -EHTs (D) stained with antibodies directed against a-actinin 2 and cTnT. Quantification of a-actinin 2 protein levels in single samples, normalized to cTnT and related to Ctrl, is depicted [n = number of samples/differentiations; 2D: Ctrl (n = 4/3); HCM (n = 4/3); HCMrep (n = 4/2); EHT: Ctrl (n = 4/2); HCM (n = 4/3); HCMrep (n = 3/3)]. Data are expressed as mean ± SEM, one-way ANOVA with Bonferroni's post-test. Abbreviations used are as follows: CMs, cardiomyocytes; cTnT, cardiac troponin T; Ctrl, control; EHTs, engineered heart tissues; MW, molecular weight marker. Source data are available online for this figure.

Fig 3: LV apical cardiomyocytes (CM) of post-P6 hearts proliferate in response to exogenous T3 versus vehicle. (A,B) Effects of T3 on DNA synthesis (EdU labeling) (A) and mitosis (pH3 labeling) (B) in cardiomyocytes from LV apex (a) or base (b). Cardiomyocytes were identified by cTnT labeling. *P < 0.05 for vehicle (Veh) versus T3 comparisons within each region using an unpaired Student’s t-test; n = 4. Intra-LV comparisons were made using a paired Student’s t-test; P-values for these comparisons are indicated. Between 500 and 2000 cardiomyocytes were analyzed for each assessment. (C) Multicolor labeling of post-neonatal mouse LV cardiomyocytes. Limited 4-hydroxytamoxifen-induced recombination of paired loxP sites (black triangles) leads to expression of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) or cyan fluorescent protein (CFP) in a few cardiomyocytes, randomly positioned within the entire LV. Insets show examples of monochromatic clusters. Intra-LV comparisons were made using a paired Students t-test. Schematics illustrate experimental protocol. Error bars indicate SEM. White bars in photomicrographs represent 20 µm.