Fig 2: Homozygous D63H human iPSCs fail to differentiate into functional cardiomyocytes and NPCs. (A, top panel) Schematic showing the differentiation from iPSCs into cardiomyocytes. (Bottom panels) Flow cytometry analysis of day 16 cardiomyocytes stained for cTnT-FITC. (B) Day 3 (top panel) and day 7 (bottom panel) iPSC-derived cardiomyocyte expression for core pluripotent genes and cardiac sarcomeric genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived cardiomyocytes from a heterozygous mother. (C) Immunofluorescence staining of day 9 iPSC-derived NPCs for Nestin, Sox2, and DAPI (10×). Images are representative of two clones for each genotype. (D) Day 9 (top panel) and day 15 (bottom panel) iPSC-derived NPC expression for core pluripotent genes assessed by qRT–PCR analysis. Data are expressed relative to iPSC-derived NPCs from a heterozygous mother.

Fig 3: D63H mutant mESCs fail to differentiate into functional cardiomyocyte foci and retain pluripotent gene expression. (A, top panel) Schematic showing the differentiation from mESCs into cardiomyocytes. (Bottom panel) Western blot analysis on bulk chromatin for Oct4 and Ac-H3K56 in wild-type versus SIRT6 knockout ESC-derived cardiomyocytes with doxycycline-inducible overexpression of wild-type SIRT6 and SIRT6 D63H. (B) Immunofluorescence staining for cTnT in mESC-derived cardiomyocytes (20×). (C) Cardiac marker expression in wild-type versus SIRT6 knockout differentiated cardiomyocytes with doxycycline-inducible overexpression of wild-type SIRT6 and SIRT6 D63H assessed by qRT–PCR analysis. Data are expressed relative to knockout cardiomyocytes. (D) Spontaneous contractile foci were quantified on day 21 as the average number of foci from five clones.

Fig 4: Loss of miR-1/133a facilitates cell cycle reentry of cardiomyocytes.(A and B) GSEA of microarray data from adult mouse hearts showing enrichment of genes related to cell cycle phase transition (A) and cell division (B) after inactivation of miR-1/133a. UO126 treatment or additional knockout of Osmr (tKO OR) or Fgfr1 (tKO FR1) abolished enrichment. Heatmaps of the 10 most up-regulated genes in dKO hearts derived from the respective gene sets confirm normalization of gene expression [ctrl: n = 4; dKO, dKO + UO, tKO (OR), tKO (FR1): n = 3]. (C and D) EdU incorporation in an isolated cTNT+ adult miR-1/133a mutant cardiomyocyte and quantification of EdU incorporation in cardiomyocytes of dKO, tKO, and control mice after EdU administration for 7 days before isolation [ctrl, n = 13; dKO, n = 10; tKO (OR), n = 14; tKO (FR1), n = 11 animals; one-way analysis of variance (ANOVA)]. Scale bars, 20 µm. (E and F) Electron microscope images of adult cardiomyocytes (ctrl, n = 3; dKO, n = 2). Scale bars, 1 µm. Sarcomeres of dKO cardiomyocytes show fuzzy Z-discs (arrowhead in inlet) and signs of disorganization. (G and H) qRT-PCR analysis of cell cycle markers in isolated adult dKO (n = 7 to 9) and control cardiomyocytes (n = 8 to 12) and noncardiomyocytes (ctrl non-CM, n = 6 to 7; dKO, n = 6 to 7; one-tailed unpaired t test). *P < 0.05 and **P < 0.01 compared to dKO; ##P < 0.01 and ###P < 0.001 compared to WT.

Fig 5: SORBS2 has a dual role in cardiogenesis.(A) Flow cytometry analysis of cardiomyocytes at differentiation day 15 (D15). P3 indicates cTnT+ population. (B) Quantification of cTnT+ cells (n = 3). **p<0.01; two-tailed Student’s t test. (C) Immunostaining of D30 cells with anti-cardiac troponin I (cTnI, red) and anti-a-actinin (green) antibodies. Boxed areas are magnified in the lower panels. (D) Quantification of cardiomyocytes with well-organized sarcomeres (control: n = 211, SORBS2-knockdown: n = 197). **p<0.01; two-tailed Student’s t test. (E) qPCR quantification of SORBS2 expression dynamics (n = 3 for each time point). **p<0.01; two-tailed Student’s t test. (F–H) qPCR quantification of second heart field (SHF) progenitor marker expression at different time points (n = 3 for each time point). *p<0.05, **p<0.01; two-tailed Student’s t test. (I) Volcano plot illustrates the differential gene expression from D5 RNA-seq data. Pink, down-regulated genes. Blue, up-regulated genes. (|log2(fold change)|>1 and padj <0.05). FC, fold change. (J, K) Gene ontology (GO) analysis of differentially expressed genes. Up-regulated pathways (J). Down-regulated pathways (K). DEGs, differentially expressed genes. (L) Heatmap illustrating gene expression changes of critical signaling pathways. Color tints correspond to expression levels. *padj <0.05. **padj <0.01. ***padj <0.001. (M) Western blot quantification of c-ABL expression on D5 cell lyses (n = 3). *p<0.05; two-tailed Student’s t test. (N) Western blot quantification of NOTCH1 expression on D5 cell lyses (n = 3). **p<0.01; two-tailed Student’s t test. (O) qPCR quantification analyses of SHH signaling target genes and SHF marker expression at D5 (n = 3 for each group). *p<0.05, **p<0.01; two-tailed Student’s t test. (P) Representative images of immunofluorescent staining of D15 cells with anti-cardiac troponin T(cTnT, green) antibody. (Q) Quantification of cTnT+ cells (n = 6). **p<0.01; two-tailed Student’s t test.