ESCs were transfected with RNAi max (Thermo Fisher Scientific) added to LSD1 siRNA (100 nM, Santa Cruz Biotechnology) or treated with GSK LSD1 at 5 μM.

LSD1 activity was performed using the EpiQuick histone demethylase LSD1 Activity/Inhibition Assay Kit from Epigentek (P-3076-48).

Total RNAs were extracted from ESCs using a Zymo Research kit. One microgram of RNA was reverse transcribed using the SuperscriptII reverse transcriptase (Invitrogen) and oligo(16)dT. Quantitative PCR was performed using LightCycler LC480 (Roche). Amplification was carried out as recommended by the manufacturer. The 12 μL reaction mixture contained 10 μL of LightCycler-DNA Master SYBR Green I mix (FAST Start KIT, containing Taq DNA polymerase, reaction buffer, deoxynucleoside trisphosphate mix, and SYBR Green I dye), 3 mM MgCl2, 0.5 μM concentration of appropriate primer, and 2 μL of cDNA. The data were normalized by PCR analysis of ATP50 expression. Results were expressed as a function of the level of expression of the gene of interest in control undifferentiated stem cells using a previously described mathematical model (52). The amplification program included the initial denaturation step at 95°C for 8 minutes, and 40 cycles of denaturation at 95°C for 3 seconds, annealing at 65°C for 10 seconds, and extension at 72°C for 10 seconds. The temperature transition rate was 20°C/s. Fluorescence was measured at the end of each extension step. After amplification, a melting curve was acquired by heating the product at 20°C/s to 95°C, cooling it at 20°C/s to 70°C, keeping it at 70°C for 20 seconds, and then slowly heating it at 0.1°C/s to 95°C. Fluorescence was measured through the slow heating phase. Melting curves were used to determine the specificity of PCR products, which were confirmed using conventional gel electrophoresis. Primers used in this study are listed in Table 1.

Heterozygous p.H222P/+ and homozygous p.H222P/H222P E13.5 embryos were collected from pregnant heterozygous p.H222P/+ females crossed with homozygous p.H222P/H222P males. Embryos were genotyped using the yolk sac. The hearts were dissected, and RNA was extracted using a Zymo Research kit. RNA was then used to generate a sequencing library or in real-time qPCR. RNA was sequenced using a NextSeq (Illumina). Sequence alignment using the mouse GRCm38/mm10 genome reached 94.91% and included 97.95% of intragenic regions. The data were analyzed using the DESeq2 application. Data are available in NCBI’s Gene Expression Omnibus database (GEO GSE156975).

Protein extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Invitrogen). Blocking and antibody incubations were performed in 5% BSA. Membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature. Antibody detection reactions were developed by enhanced chemiluminescence (MilliporeSigma).

ESCs or EBs were fixed in 3% paraformaldehyde for 15 minutes at room temperature. After blocking in PBS-BSA 3% 0.1 % Triton X-100 cells were incubated in PBS, 0.01 % Triton X-100, 3% BSA with the following: mouse anti-SSEA1 (Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA, clone MC480), rabbit anti–Oct-3/4 antibody (Santa Cruz Biotechnology, catalog H134), mouse anti-sarcomeric α−actinin antibody (51) (1/500, Sigma-Aldrich, catalog H7811), anti-Nkx2.5 antibody (Santa Cruz Biotechnology, catalog H114), anti-Isl1 antibody (Hybridoma Bank, clone 39.3F7), anti-Mef2c antibody (Aviva Systems Biology, catalog ARP37342_T100), anti-LSD1 (Abcam, catalog ab17721), and anti-βMHC antibody (Alexis Biochemicals, clone F109.3E1). Cells were then incubated with the appropriate secondary anti–rabbit or anti–mouse IgG coupled to Alexa Fluor 488 or Alexa Fluor 546 conjugated antibodies (Molecular Probes, Life Technologies, Thermo Fisher Scientific) diluted to 1/500 in PBS, 0.1% Tween 20, 10% donkey serum. Cells were incubated for 5 minutes in DAPI (Sigma-Aldrich) before mounting (Fluoromount-G, SouthernBiotech). Images were acquired using an apotome (Zeiss) and deconvoluted using AutoQuant software (Bitplane).

EBs were loaded with 50 μM Fluo-A in cell culture medium from a 1 mg/mL stock in DMSO/20% Pluronic acid Fluo4-AM (Thermo Fisher Scientific) for 15 minutes at 37°C. Ca2+ transients at 37°C in DMEM, 10% FCS, and 10 mM HEPES were measured with a Zeiss observer microscope equipped with a Hammatsu Camera (2048 × 2048 pixels, 100 frames/s), using Zen-Pro software. Regions of interest were selected in cells within a beating area of EBs, and the average pixel intensity was plotted as a function of time. All experiments were performed at 35°C ± 2°C. The fluorescence signal was expressed as F-F0/F0, F0 being the lowest fluorescence signal recorded.

ChIP was performed according to previously reported protocols (53). The ChIP-grade anti-H3K4me1 (c-15210018), anti-H3K27ac (c-15210016), and anti-H3K9me2 (c-15200154) antibodies were from Diagenode. qPCR primers for ChIP assays are listed in Table 2.

Staining of heart sections was performed as previously described (54). Briefly, embryos or adult hearts were fixed overnight at 4°C with 4% paraformaldehyde, washed with PBS. The samples were dehydrated using ethanol 50%, 70%, and 100% and then xylene prior to paraffin embedding. The embryos were cut with a microtome. After rehydration, heart sections were stained with H&E or Sirius red, or permeabilized with PBS 0.1% Triton X-100 and blocked with PBS 0.1% Tween 20 with 10% donkey serum; mouse anti-sarcomeric α−actinin antibody (1/500, Sigma-Aldrich, catalog H7811), was added in PBS 10% donkey serum and 0.1% Tween 20. The secondary antibody Alexa Fluor 488 was added in PBS 0.1% Tween 20.

Histopathological analysis of embryonic hearts was performed using ImageJ software (NIH).

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