Fig 2: cP1P-augmented cardiac differentiation in hESCs is dependent on BMP receptor-mediated SMAD1/5/8 signaling. (A) The activity of BMPR/SMAD signaling with or without cP1P treatment was evaluated by the expression levels of ALK2 and ALK3 by Western blot at the indicated time points along with GAPDH expression as the loading control. (B) Schematic representation of the treatment with BMPR inhibitor LDN during days 3 to 5 of CM differentiation coinciding with IWR1 inhibitor treatment. (C) The activity of BMPR/SMAD signaling was inhibited by the administration of LDN, as shown by the downregulation of ALK3 and p-SMAD 1/5/8, while cP1P treatment could not reverse the inhibitory effect of LDN. The effects of BMPR inhibition by LDN on cardiomyocyte differentiation, shown by (D) the downregulation of NKX2.5 at day 5. (E) Treatment of LDN between day 3 to 5 also downregulated the expression of TNNT2 and MLC2V at day 8 of CM differentiation even upon cP1P treatment, as demonstrated by Western blot. (F) Schematic representation of the treatment with LDN during days 5 to 8 of CM differentiation. (G) Immunoblots of ALK3, p-SMAD1/5/8, SMAD1/5/8, and NKX2.5 at day 5 of LDN treatment in DMSO-treated controls and cP1P-treated CMs. (H) Immunoblots of TNNT2 and MLC2V at day 8 of LDN treatment in DMSO-treated controls and cP1P-treated CMs.

Fig 3: Generation and validation of TNNT2-FUCCI in hiPSC-derived CMs. (A) Schematic representation showing TNNT2-FUCCI reporter cell line generation and differentiation towards cardiomyocytes. Upon differentiation into cardiomyocytes, the FUCCI signal became visible at Day 6. Arrowheads indicate FUCCI+ nuclei. Scale bar, 20 µm. (B) Immunocytochemistry showing cardiomyocytes at Day 30 of differentiation. Arrowheads indicate cardiomyocytes (TNNT2+) that were FUCCI+, demonstrating the specificity of the FUCCI reporter. Scale bar, 20 µm. (C) Imaging flow cytometry shows TNNT2+ cells expressing nuclear mVenus and/or mCherry, indicating cardiomyocyte specificity of the TNNT2-FUCCI system.

Fig 4: The increasing expression of LKB1 inhibits proliferation of NRVMs. (A) The relative mRNA and protein expressions of LKB1 in the heart at different postnatal time point are shown. Relative mRNA levels of Lkb1 in P1, P7, and P28 mice hearts were assayed by qRT-PCR (n = 5) in A1; (A2) Relative protein expression of LKB1 in P1, P7, and P28 mice hearts were detected by western blots (n = 8); (A3) The quantitative grayscale density of western blots of A2; GAPDH was used for normalization; (B) Representative confocal microscopy of Ki67 and pH3 immunostaining at P7 and P28 hearts (Ki67+, pH3+ in red, and cTnT+ in green indicate proliferating cardiomyocytes). The Ki67 and pH3 positive cell percentages (from total >1000 cells) were calculated and shown in B2, scale bar = 50 µm. (C) Confocal microscopy of cell proliferation of NRVMs with LKB1 overexpression (Ki67+, pH3+, and Aurora B+ in red, and cTnT+ in green indicate proliferating cardiomyocytes), quantified in C2. 2000–5000 cells were randomly selected and quantified in each group, scale bar = 20 µm; (D) Cell cycle-associated gene expressions of NRVMs treated with vector or LKB1 overexpression plasmid were detected by qRT-PCR; (E) Representative images (E1) of cell cycle-related protein expression and quantification in E2, were analyzed by western blots (n = 6, GAPDH was used as internal control), *p < 0.05, **p < 0.01, ***p < 0.001. p: Postnatal day; NRVMs: Neonatal rat ventricular myocytes; LKB1: Liver kinase B1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR: Quantitative real-time PCR.

Fig 5: Neonatal hyperoxia represses fatty acid synthesis enzymes in murine atrial cardiomyocytes.(A, B, D, and E). Sections of left atrial appendages from P4 neonates exposed to room air (A and B) or hyperoxia (D and E) costained for FASN (red, A and B) or SCD1 (red, D and E) and TNNT2 (green, A, B, D, and E). Sections were also stained with DAPI to label nuclei (blue, A, B, D, and E). Arrows and arrowheads show TNNT2+ cardiomyocytes in left atrial appendage and LV, respectively. Scale bars: 200 µm. (C and F) Graphs show relative staining intensities for Fasn (C) and Scd1 (F) measured using NIH ImageJ 2.0/Fiji. (C and F) Room air, n = 5; hyperoxia, n = 5. Box plots show median values and inner quartiles. Whiskers show the range of values. Circles show values for individual room air– and hyperoxia-exposed mice, respectively. F tests were used to determine if samples had equal or unequal variances. P values are the results of unpaired 2-tailed t tests.