MA5-12960

antibody from Invitrogen Antibodies

Targeting: TNNT2 CMD1D, CMH2, CMPD2

Provider product page for MA5-12960

Western blot

Immunocytochemistry

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Antibody data

Antibody Data
Antigen structure
References [214]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [3]
Other assay [100]

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Product number: MA5-12960 - Provider product page
Provider: Invitrogen Antibodies
Product name: Cardiac Troponin T Monoclonal Antibody (13-11)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA5-12960 targets Troponin T Cardiac Isoform in IF/ICC, IHC (P), and IM applications and shows reactivity with Avian, Canine, Chicken, Fish, Guinea Pig, Human, mouse, Porcine, Rabbit, and Rat samples.
Antibody clone number: 13-11
Concentration: 0.5 mg/mL

TNNT2 protein structure - MA5-12960 shown in red.

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Experimental details: Fig 4 Expression and phosphorylation of AS160 and TBC1D1 in the infarct heart. Immunoblotting analyses of phosphorylation of AS160-Thr 649 and PKB-Ser 473 , and total AS160, TBC1D1, PKB, GLUT4 and cTnT in the lysates (40 mug) of the infarct zone (I), border zone (B) and remote zone (R) of mouse heart 4 weeks after myocardial infarction. Flotillin-1 was used as a loading control. A, representative blots. B, quantitation of TBC1D1 signals, n = 4. Asterisk indicates p < 0.05 (infarct zone versus remote zone).

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Experimental details: Figure 4 hPSC-derived Cardiomyocyte Proliferation Screening in the HDMA Platform. ( A ) Experimental timeline used in the screen. ( B ) Representative flow cytometric analyses of cell population used in HDMA screening, with percentages marked. Dot plots of CD90 + stromal cell content from 15 d digestion of differentiated hPSCs; dot plots of Ki67 and cTnT expression in both 15 d digestion of differentiated hPSCs and cells replated for 2 d prior to assay startpoint. ( C ) Representative phase contrast micrographs of CMs before and after seeding, and cultured for up to 3 d with drug treatments, as indicated. Scale bars for indicated objective magnification: 4x, 200 mum; 10x, 200 mum; 20x, 100 mum. ( D ) Matrix of design culture conditions in each of the 81 column pairs in the array, for the screened factors: CHIR99021 (CHIR), muM; Purmorphamine (Pm), muM; IGF-1, ng/mL; FGF-2, ng/mL. DMSO was included so as to be at a uniform total concentration of 0.05% v/v in the CHIR channels, acting as a vehicle control. ( E ) Tile-scan confocal image of the entire HDMA (~91 x 28 mm) treated for 24 h and used in further analysis, after fixation and in situ immunostaining for Ki67, cTnT and DNA. ( F ) Higher-magnification confocal image of subsection of replicate HDMA treated for 3 d, showing cell population arrangement in arrayed culture chambers. ( G ) Confocal image showing detail of individual HDMA chambers from replicate HDMA treated for 3 d, demonstrating presence of various proliferatin

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Experimental details: Figure 6 Confirmation of HDMA Screen Results in Standard Static Cultures. ( A ) Confocal fluorescence images of Ki67 expression in myocyte population after 24 h treatment with Control medium or medium containing 5 muM CHIR. Scale bar: 100 mum. ( B ) Scatterplot of results from HDMA individual column-pairs against conditions tested in standard static culture, with linear regression and correlation coefficient marked. ( C ) Quantification of percentage Ki67 + myocytes after 24 h treatment in static cultures. Bars represent mean +- S.D. of 2-3 independent experiments including separate cardiomyocyte inductions. *indicates p < 0.05 by one-way ANOVA with Tukey post-hoc tests vs. None condition. No other differences were significant. p -values (paired two-tailed t -test) against None condition are indicated for treatments with large effect magnitudes. ( D ) Quantification of total myocytes after treatment for 3 days with Control (None) medium or medium containing 5 muM CHIR. Bars represent mean +- S.D. of 4 independent experiments including inductions, normalised to control (None) condition. p -value (paired two-tailed t -test) indicated. ( E ) Quantification of percentage of binucleated cardiomyocytes after treatment for 3 days with Control (DMSO 0.05% v/v) medium or medium containing 5 muM CHIR. Dotplot from flow cytometric analysis of nuclear staining (Hoechst 33342) pulsewidth versus pulse area. Gating is on singlets by FSC and SSC area, height and width, and cTnT + cardiomyocy

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Experimental details: Figure 1 Differentiation and Purification of hiPSC-derived CMs (A) Sequencing confirmed the heterozygous BRAF T599R (BRAF1) and the Q257R mutations (BRAF2, BRAF3) in hiPSCs. Red asterisks note the positions of the BRAF mutations. (B) Cardiac differentiation protocol. hiPSCs were exposed to a series of cytokines to induce cardiogenesis. CMs were sorted for purification after day 25. (C) Cell-sorting strategy to purify SIRPalpha + /CD90 - cells and SIRPalpha - /CD90 + cells. (D) Staining for cTNT demonstrated purification of >95% CMs after sorting for SIRPalpha + /CD90 - cells. See also Figures S1 and S2 .

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Experimental details: Figure 5 BRAF-Mutant FLCs Influence the Hypertrophic Phenotype (A) Schematic of co-culture experiment. WT CMs and WT and BRAF-mutant FLCs (Fib-like) were sorted from EBs and re-cultured together. (B) Representative images of co-culture treatment groups stained with cTNT. Scale bars, 200 mum. (C) Quantification of cellular area as depicted in (B). WT CMs became significantly larger upon co-culture with BRAF-mutant Fib-like cells (n = 67) compared with co-culture with WT Fib-like cells (n = 62) ( **** p < 0.0001). Box-and-whisker plots show the median to the first and third quartiles and the minimum and maximum values. Data represent two biological (WT2, 3; BRAF1, 2) and three technical replicates. (D) Increased proliferation rate of purified BRAF-mutant Fib-like cells (n = 3) compared with WT (n = 2) as demonstrated by increased staining for Ki67 by flow cytometry ( * p = 0.03). Data represent two (WT2, 3) or three (BRAF1, 2, 3) biological and two technical replicates. Data are presented as means +- SEM. (E) Purified BRAF-mutant Fib-like cells expressed increased levels of fibrosis-associated genes compared with WT; TGFbeta1 (BRAF n = 24, WT n = 18, ** p = 0.002), POSTN (BRAF n = 15, WT n = 3, * p = 0.01), CTGF (BRAF n = 18, WT n = 12, ** p = 0.002), COL1A2 (BRAF n = 24, WT n = 15, ** p = 0.003), and ET-1 (BRAF n = 15, WT n = 8, p = 0.07, not significant). Data represent three biological (WT1, 2, 3; BRAF1, 2, 3) and three technical replicates. Data are presented as means +- SE

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Experimental details: Figure 6 BRAF-Mutant FLCs Influence CM Hypertrophy through Paracrine TGFbeta1 Secretion (A) Schematic of conditioned media experiment. WT and BRAF-mutant CMs and FLCs (Fib-like) were sorted from EBs and re-cultured separately. After 4 days, CMs were exposed to Fib-like conditioned media. (B) Representative images of treatment groups stained with cTNT. TGFbeta-NA indicates pre-incubation of Fib-like conditioned media with a TGFbeta neutralizing antibody prior to CM exposure. Scale bars, 200 mum. (C) Quantification of cellular area as depicted in (B). WT CMs exposed to BRAF-mutant Fib-like conditioned media (n = 97) became significantly enlarged compared with exposure to WT Fib-like conditioned media (n = 91) ( **** p < 0.0001). This effect was prevented by pre-incubation with TGFbeta-NA (n = 92). Cellular hypertrophy in BRAF-mutant CMs (n = 86) was significantly reduced upon incubation with WT Fib-like conditioned media (n = 88) ( ** p = 0.008), or pre-incubation of BRAF-mutant Fib-like conditioned media with TGFbeta-NA (n = 134) ( * p = 0.01). Box-and-whisker plots show the median to the first and third quartiles and the minimum and maximum values. ns, not significant. (D) WT CMs exposed to BRAF-mutant Fib-like conditioned media upregulated ANP (n = 3) compared with WT Fib-like conditioned media (n = 3) ( * p = 0.03). Data are presented as means +- SEM. (E) BRAF-mutant CMs exposed to BRAF-mutant Fib-like conditioned media (n = 3) upregulated BNP expression ( ** p = 0.001), wh

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Experimental details: Figure 1 High purity of cardiomyocytes can be obtained under glucose-depleted and fatty acid (with or without T 3 )-supplemented conditions. (A) The schematic for purification and maturation using metabolic selection. Glycogen synthase kinase 3 inhibitor CHIR99021 and Wnt pathway inhibitor IWP2 (gray boxes) were added to the differentiation medium on differentiation days 1 and 4, respectively. On differentiation day 10, cells were dissociated and plated on glass cover slips. After 3 days recovery, cells were cultured with metabolic selection medium (No-glucose DMEM medium supplemented with lactate, fatty acid, and fatty acid + T 3 ). On differentiation day 18, cells were collected for patch clamping experiments. On differentiation day 21, cells were collected for other experiments. (B) Representative fluorescence-activated cell sorting (FACS) analyses of cardiac Troponin T expression in the human pluripotent stem cell (hPSC)-derived cells on day 0 with routine culture medium, and on day 8 with metabolic selection medium. In the control group, the isotype control (mouse IgG) was used instead of the primary antibody. (C) Time courses of selection efficiency using the lactate-supplemented condition (red), the fatty acid-supplemented condition (green), and the fatty acid with T 3 -supplemented condition (purple); cells cultured with routine medium (blue) were set up as the control groups ( n = 3). The asterisk means each test group is significantly different from controls, P < 0.

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Experimental details: Development of titin, myosin heavy chain (MyHC) and troponin T (TnT) isoform expression. A, Representative Coomassie-stained 1% SDS-agarose gel of titin isoforms in RV samples obtained at the indicated ages; rabbit soleus and rat left ventricle (LV) were used as markers. B, Western blot probing for TnT, guinea pig myocardium expressing 4 isoforms, and human recombinant (hcTnT) are shown for comparison; in all human samples the predominant isoform is TnT3. C, Representative SDS-PAGE of separation of MyHC isoforms, markers for alpha- and beta-MyHC isoforms: atrium (A), nonfailing adult heart (NF), and comigration (NF:A). Pediatric patients express only the beta-MyHC.

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Experimental details: Figure 3 FIGURE 3: Mitochondrial functions increase during cardiomyoblast differentiation. Protein levels and immunofluorescence staining of myosin heavy chain (MHC) (A) and cardiac Troponin T (B) were performed in H9c2 cells following differentiation for 0, 2, 4 and 7 days. HSP60 (C) , PDH (D) , PGC1alpha (E) protein levels and citrate synthase activity (F) were determined during this period. (G) Oxygen consumption rate (OCR) was measured in H9c2 cells following 7 days differentiation. (H-I) HIF1alpha, Hexokinase 2 (HK2), NIX and BNIP3 protein levels were examined and quantified in H9c2 cells during the indicated days of differentiation. alpha-tubulin was used as a loading control. All quantitative data are mean +- SEM from 3 independent experiments. Scale bar, 20 mum. * P < 0.05.

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Experimental details: Figure 5 FIGURE 5: Involvement of HIF1alpha in mitophagy and cardiomyocyte differentiation. H9c2 cells were cultured in differentiation medium for 7 days. (A) Representative images of mito -QC H9c2 cells transfected with non-targeting (NT) siRNA or HIF1alpha siRNA at day 4 of differentiation. Mitophagy mask represented as the mCherry/GFP ratio intensity above the mean of mCherry intensity. (B) Quantitation from (A) of total mitolysosome area per mitochondrial content as indicated. (C) OCR was measured following 7 days H9c2 differentiation with cells transfected with NT siRNA or HIF1alpha siRNA at day 4. 1 muM oligomycin A, 1 muM FCCP and 1/2 muM rotenone/antimycin A were injected at the indicated times to determine the proportion of oxygen consumption due to ATP turnover, maximal rate of respiration and amount of proton leak respectively. (D-E) HIF1alpha, MHC and cardiac Troponin T protein levels were examined in 7 days-differentiated H9c2 cells transfected with NT or HIF1alpha siRNA at day 4. alpha-tubulin was used as a loading control. Scale bar, 20 mum. All quantitative data are mean +- SEM from 3 independent experiments. * P < 0.05.

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Experimental details: Figure 6 FIGURE 6: NIX-mediated mitophagy is not essential for cardiomyocyte differentiation. H9c2 cells were cultured in differentiation medium for 7 days. (A) Representative images of mito -QC H9c2 cells transfected with non-targeting (NT), BNIP3, or NIX siRNA as well as NIX and BNIP3 (NIX/BNIP3) siRNA in combination at day 4 differentiation. Mitophagy mask represented as the mCherry/GFP ratio intensity above the mean of mCherry intensity. (B) Quantitation from (A) of total mitolysosome area per mitochondrial content as indicated. (C-D) BNIP3, NIX, MHC and cardiac Troponin T protein levels were examined after H9c2 cells transfected with NT, BNIP3, NIX or NIX/BNIP3 siRNAs at day 4 differentiation. Vinculin was used as a loading control. (E) H9c2 cells stably expressing WT or siRNA resistant NIX were cultured in differentiation medium for 7 days. NT siRNA or NIX siRNA was applied into medium at day 4 differentiation. NIX, MHC and cardiac Troponin T protein levels were examined after 7 days differentiation. Quantitation from (E) of indicated proteins was shown in (F) . alpha-tubulin was used as a loading control. (G) Representative images of NIX WT or siRNA-resistant cells transfected with NT siRNA or NIX siRNA at day 4. (H) Quantitation from (G) of total mitolysosome area per mitochondrial content as indicated. Scale bar, 20 mum. All quantitative data are mean +- SEM from 3 independent experiments. * P < 0.05.

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Experimental details: Figure 7 FIGURE 7: Increasing NIX-dependent mitophagy promotes cardiomyocyte differentiation. (A) Representative images from mito -QC H9c2 vector control cells or cells overexpressing NIX cultured following 7 days differentiation. Mitophagy mask represented as the mCherry/GFP ratio intensity above the mean of mCherry intensity. (B) Quantitation from (A) of total mitolysosome area per mitochondrial content as indicated. (C) Representative immunoblot of NIX, BNIP3, MHC and cardiac Troponin T protein levels from 7 days differentiated H9c2 cells stably overexpressing NIX. (D) Quantitation of data from (C). (E) Representative images from mito -QC H9c2 cells overexpressing vector or BNIP3 cultured in differentiation medium for 7 days. (F) Quantitation from (E) of total mitolysosome area per mitochondrial content as indicated. (G) Representative immunoblot of NIX, BNIP3, MHC and cardiac Troponin T protein levels from 7 days differentiated H9c2 cells stably overexpressing BNIP3. (H) Quantitation of data from (G). (I) Oxygen consumption rate (OCR) was measured after H9c2 cells stably overexpressing vector or NIX following 7 days differentiation. (J) Immunoblot of HK2 and Pyruvate kinase (PKM2) from H9c2 cells stably overexpressing vector or NIX cultured in differentiation medium for 7 days. Quanitation of HK2 (K) and PKM2 (L) is shown. Scale bar, 20 mum. All quantitative data are mean +- SEM from 3 independent experiments. * P < 0.05.

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Experimental details: Figure 1 Induced Alternative Reading Frame (ARF) expression in hs :ARF fish suppresses cardiac regeneration. ( a ) Representative polymerase chain reaction (PCR) product of ARF expression from three experimental replicates of wild type (WT) and hs :ARF heart RNA on 15 dpi. The product run on gel electrophoresis shows ARF expression present in hs :ARF fish while absent in WT fish. ( b ) Acid Fuchsin Orange G (AFOG) and troponin staining of WT and hs :ARF heart cryosections on 15 days post-injury (dpi). The dotted lines demarcate the injury site. Red stains are for fibrin. Blue stains are for collagen. Brown stains are for muscle. Green stains are for troponin. ( c ) Myocardial recovery measured by troponin infiltration into the injury site. Infiltration quantified by imaging software shows decreased troponin in the injury site of hs :ARF fish compared to WT fish. N = 8 hearts. Results are shown as mean +- standard error. The * represents a statistically significant difference.

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Experimental details: Figure 2 ARF expressed under control of the endogenous human ARF promoter in ARF :ARF fish suppresses cardiac regeneration over time. ( a ) AFOG and troponin staining of WT and ARF :ARF heart cryosections at 1, 7, 15, and 30 dpi. The dotted lines demarcate the injury site. Red stains are for fibrin. Blue stains are for collagen. Brown stains are for muscle. Green stains are for troponin. ( b ) Myocardial recovery measured by troponin infiltration into the injury site. Infiltration quantified by imaging software shows decreased troponin in the injury site of ARF :ARF fish compared to WT fish from 7 dpi onward. N = 49 hearts. The grey and black lines are logarithmic approximations of the WT and ARF :ARF heart recovery trends respectively. Results are shown as mean +- standard error. The * represents a statistically significant difference.

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Experimental details: FIGURE 2 The proliferating ventricular cardiomyocytes were efficiently labeled in Tnnt2-Dre x Aurkb-rox-tdTomato neonates. (A) Tnnt2-Dre mice were used to cross with Aurkb-rox-tdTomato mice to label proliferating cardiomyocytes. (B) Immunostaining showed the coexpression of tdTomato with cTnT, PECAM, a-SMA, or Col1 on P1 Tnnt2-Dre x Aurkb-rox-tdTomato heart sections. Scale bar = 40 mum. (C) Immunostaining for tdTomato with Aurkb on P56 Tnnt2-Dre x Aurkb-rox-tdTomato heart sections. Scale bar = 20 mum. (D) Representative images of flow cytometry analysis (D1) and quantification of tdTomato +- cells (D2) and numbers in G1, S, G2/M phases (D3) from ventricles of P1 Tnnt2-Dre x Aurkb-rox-tdTomato mice. (D2) n = 6, * p < 0.05 vs. tdTomato - cTnT - ; # p < 0.05 vs. tdTomato + cTnT - ; & p < 0.05 vs. tdTomato - cTnT + . (D3) n = 5, * p < 0.05 vs. tdTomato - cTnT + . (E) Representative images (E1) and quantification (E2) of time-lapse imaging analysis showed cell proliferation in vitro of tdTomato + cardiomyocytes from the ventricles of P1 Tnnt2-Dre x Aurkb-rox-tdTomato mice. Scale bar = 20 mum.

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Experimental details: Fig. 7 a Representative confocal microscope images of hiPSC aggregates taken from the final day of the vertical-wheel bioreactor serial passage (day 12) and stained for pluripotency markers SSEA-4, TRA-1-60, and Nanog (scale bar = 100 mum) and b differentiated into neuronal cells (immuno-stained for beta-tubulin and PAX6), hepatocytes (immuno-stained for HNF4alpha), and cardiomyocytes (immuno-stained for TNNT2) (scale bar = 50 mum)

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Experimental details: Figure 3 The mitochondrial distribution is similar in both 3D cardiac spheroids differentiated under LOE and HOE conditions. Mitochondrial localization and cell area in 25-day-old hPSC-CMs (using hiPSC UEFhfiPS1.4 and hESC CCTL12 lines) differentiated in LOE and HOE conditions were evaluated using MitoTracker dye. ( A ) Immunostaining showing the MitoTracker signal (red) and the cTnT staining (green) in 25-day-old hPSC-CMs obtained from 3D cardiac spheroids differentiated under LOE and HOE conditions. Nuclei are stained with DAPI (blue). Scale bars: 40 um. ( B ) Mitochondrial mean distance in 25-day-old hPSC-CMs obtained from 3D cardiac spheroids differentiated under LOE (white dot plots) and HOE (black dot plots) conditions. ( C ) Mitochondrial maximal distance in 25-day-old hPSC-CMs obtained from 3D cardiac spheroids differentiated in LOE and HOE conditions. ( D ) Cell area of 25-day-old hPSC-CMs (using hiPSC UEFhfiPS1.4 and UB47 lines) obtained from 3D cardiac spheroids differentiated in LOE and HOE conditions. ( E ) DeltaDeltaCt relative gene expression analysis by qRT-PCR in 25-day-old hPSC-CMs obtained from 3D cardiac spheroids differentiated in LOE (white bars) and HOE (black bars) conditions for BNP= natriuretic peptide B and ACTA1= actin alpha skeletal muscle 1. ( F ) Measurement of the sarcomere length of hPSC-CMs (using hiPSC UEFhfiPS1.4 and hESC CCTL12 lines) in the two differentiation protocols by tracing a line of 17 uM across the sarcomeres using an in-house de

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Experimental details: Fig. 4 Fabrication of cardiac spheroids for disease modeling applications. a (i) Development of healthy and scarred spheroids through mixing iPSC-derived cardiomyocytes (iPSC-CMs) with primary adult cardiac fibroblasts (CFs) at defined ratios of cell numbers (4:1 for healthy; 1:4 for scarred). Top: Images (cardiac troponin-T (cTnT) (red; iPSC-CMs); vimentin (green; CFs)) taken 3 days after cell seeding. Scalebar 50 um. Bottom: Immunofluorescence staining for alpha-actinin (green; sarcomeres) and cTnT (red; iPSC-CMs) in healthy and scarred spheroids at 3 days. Scalebar 10 um. (ii) Quantification of cellular composition through staining for cTnT (iPSC-CMs) and vimentin (CFs) ( n = 3 biologically independent samples, mean +- s.d, two-sided student t test, healthy - cTnT + vs. Vimentin + p = 5.6 x 10 -5 , scarred - cTnT + vs. Vimentin + p = 6.7 x 10 -4 ). b (i) Contraction profiles, (ii) contraction amplitude (a.u. absolute units), and (iii) peak-to-peak time (ms) of healthy and scarred cardiac spheroids at 3 days ( n = 3 biologically independent samples, mean +- s.d, two-sided student t test, (ii) p = 5.0 x 10 -4 ). c Quantification of calcium activation parameters from calcium mapping experiments in healthy and scarred spheroids at 3 days, including (i) calcium transient duration (ms), (ii) time-to-peak (ms), and (iii) calcium flux amplitude (F/Fo) ( n = 5 biologically independent samples, mean +- s.d, two-sided student t test, (i) p = 0.0014, (ii) p = 5.0 x 10 -6 , (iii) p = 2

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Experimental details: Fig. 5 3D bioprinting cardiac microtissues for disease modeling applications. a (i) Schematic of 3D bioprinting of healthy and scarred cardiac microtissue rings and (ii) immunofluorescence staining for cTnT and vimentin in healthy and scarred cardiac microtissues after 5 days of fusion within the support hydrogel. Scalebar 100 um (insets 50 um). (iii) Immunofluorescence staining for alpha-actinin (green; sarcomeres) and cTnT (red; iPSC-CMs) and (iv) connexin-43 (green; gap junctions) and cTnT (red; iPSC-CMs), in healthy and scarred regions of microtissues after 5 days of fusion within the support hydrogel. Scalebar 10 um. b (i) Contraction profiles of healthy and scarred cardiac microtissues following removal from the support hydrogel after 5 days of culture. (ii) Contraction amplitude (a.u. absolute units) and (iii) Peak-to-peak time (ms) at 5 days ( n = 3 biologically independent samples, mean +- s.d, two-sided student t test, (ii) p = 0.061). c (i) Calcium mapping in healthy and scarred cardiac microtissues after 5 days culture, each image represents a 20 ms frame. Scalebar 100 um. (ii) Representative calcium traces from regions 1 and 2 (see methods) in healthy and scarred cardiac microtissues. Scalebars 0.5 DeltaF/F o (y), 500 ms (x). (iii) Activation maps of healthy and scarred cardiac microtissues, and activation delay (ms) (difference in activation time (ms) between regions 1 and 2) in healthy and scarred cardiac microtissues ( n = 3 biologically independent samples, m

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Experimental details: Fig. 6 Evaluating miRNA on the behavior of cardiac microtissues. a (i) Schematic of cholesterol modified miR302 (chol-miRNA 302 b/c) delivery to scarred cardiac spheroids for 0, 0-2, 0-4, and 0-7 days. (ii) Contraction amplitude (a.u) and (iii) peak-to-peak time (ms) within scarred spheroids measured after 2, 4, and 7 days for each treatment period ( n = 4, 6, 5, 5, 7, 5, 5, 5, 5 biologically independent samples (from left to right), mean +- s.d, one-way ANOVA, (ii) day 4: 0 vs. 0-4 days treatment p = 0.014, (ii) day 7: 0 vs. 0-7 days treatment p = 0.0045, (iii) day 4: 0 vs. 0-4 days treatment p = 1.6 x 10 -7 , (iii) day 7: 0 vs. 0-7 days treatment p = 4.1 x 10 -9 ). b (i) Immunofluorescence staining for cTnT (red; iPSC-CMs), vimentin (green; cardiac fibroblasts), and EdU (proliferation marker) in scarred spheroids at day 7 for each treatment condition. Top panel scalebar 50 um, and bottom panel scalebar 40 um. (ii) Quantification of cardiomyocyte proliferation (EdU + and cTnT + ) and (iii) fibroblast proliferation (EdU + and Vimentin + ) at day 7 for each treatment condition. ( n = 3, 4, 4, 4 biologically independent samples (from left to right), mean +- s.d, one-way ANOVA, 0 vs. 0-4 days treatment p = 0.044, 0 vs. 0-7 days treatment p = 0.026). Scalebar 50 um. c (i) Experimental outline where scarred microtissues are bioprinted in the support hydrogel as previously described, followed by 4 days treatment with miR302, and analysis compared to non-treated controls. (ii) Calci

Supportive validation

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Experimental details: 966.g005 Fig 5 Cardiomyocyte differentiation efficiency of AICS11 co-cultured with iCMs evaluated by flow cytometry. AICS11 were either co-cultured with IMR90-iCMs, or subjected to media change only as a control. Additional controls include non-differentiated iPS or GiWi-differentiated AICS11 or IMR90 cells. Total cells were harvested and immuno-stained for the cardiomyocyte markers (A) cTnT and (B) a-actinin, and analyzed by flow cytometry in conjunction with TOM20-GFP to distinguish AICS11 cells. As shown are representative flow plots for an experiment performed in triplicates. Calculation of cardiomyocyte differentiation efficiency for (C) cTnT and (D) a-actinin. Green bars indicate cells gated for GFP only, where % = Q2/(Q2+Q3)*100, while grey bars indicate total cells, where % = (Q1+Q2)/(Q1+Q2+Q3+Q4)*100. P-value for all pairwise comparison is

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Experimental details: 966.g006 Fig 6 Cardiomyocyte differentiation efficiency of AICS16 co-cultured with iCMs evaluated by flow cytometry. Description for this experiment is identical to that for Fig 5 , but with AICS16 cells (expressing GFP-b-actin) in place of AICS11. P-value for all pairwise comparison is

Supportive validation

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Experimental details: Figure 6 Fibroblast-secreted mediators induce 3-dimensional patterning of VCS markers. Evaluation of the influence of fibroblast-secreted factors on the expression of VCS markers in neonatal rat cardiomyocyte organoids using automated high content screening analysis. Phenotypic parameters were retrieved for each single-cell identified in 12 Z-planes and only cardiomyocytes were included in the analysis. ( A ) Illustration of cardiomyocyte organoid analysis ( B ) Total number of cells per z plane. N = 6-8. ( C - J '') Control and fCM-treated organoids representative z-stack immunofluorescence images from summed intensities. Cell nuclei (DAPI), cardiomyocytes (cardiac Troponin T positive cells, Red), connexin 40 and NAV1.5 (Green). Scale bars = 100 um. ( K - N '') Single z plane view of representative outer surface regions of control and fCM-treated organoids. ( O - P ) Mean expression of connexin 40 and NAV1.5 per cardiomyocyte given by the average of fluorescence intensity in the cardiomyocyte population. The mean fluorescence intensity was measured in three different z-positions for the organoid outer (z4-z6) and inner surfaces (z8-z10). Results are disposed as a mean +- SEM. Data were subjected to a two-way ANOVA followed by a post test comparing each group to every other group; p < 0.05 was considered significant. N = 9-12.

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Experimental details: Figure 3 HO-1 does not influence the efficiency of hiPSCs.2 differentiation to cardiomyocytes. ( A ) Immunofluorescence analysis of markers of three germ-layers (Vimentin, alpha-SMA, GATA4, AFP, and NFH) in spontaneously differentiated WT (upper panel) and HO-1 KO (bottom panel) hiPSC.3 via embryoid bodies. Bar indicates 100 um. qRT-PCR analysis of expression of cardiac mesoderm markers: ( B ) ISL1 , ( C ) GATA6 , ( D ) TNNT2 and ( E ) MIXL1. Expression was normalized to EEF-2 levels. ( F ) Flow cytometric analysis of direct cardiomyocyte differentiation efficiency (based on TNNT2 expression) of WT hiPSC.2 treated with tin protoporphyrin IX (SnPP), cobalt protoporphyrin IX (CoPP) and hemin. Ctr1-DMSO control for SnPP and CoPP, ctr2-2 uM NaOH control for hemin. Bars represent mean +- SD of N = 3 experiments. Dots, squares and triangles represent each replicate for corresponding groups. * p < 0.05, one-way ANOVA test.

Supportive validation

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Experimental details: Figure 1 Neonatal hyperoxia enlarges the left atria of mice but reduces the density of cardiomyocyte (CM) nuclei in the left atrial myocardia. ( A ) H&E-stained sections of hearts from P4 mice that were exposed to room air (left) or hyperoxia (right). Dotted lines outline the left atria. ( B ) Mean area of the left atria in sections of room air- and hyperoxia-exposed mice on P4. n = 4 mice per condition. ( C ) Numbers of DAPI-stained nuclei per mm 2 of TNNT2 + myocardium. Room air, n = 4; hyperoxia, n = 5 mice per condition. ( D and G ) Sectioned hearts of P56 ( D ) and P365 ( G ) mice exposed to room air (right) or hyperoxia (right) from P0-P4 stained with Masson's trichrome. ( E and H ) Graphs show mean area of the left atria in sections of room air- and hyperoxia-exposed mice on P56 ( E ) and P365 ( F ). ( E ) n = 4 per condition. ( H ) Room air, n = 3; hyperoxia, n = 4. ( F and I ) Graphs show numbers of DAPI-stained nuclei per mm 2 of TNNT2 + myocardium in room air- and hyperoxia-exposed mice on P56 ( F ) and P365 ( I ). ( F ) n = 3 mice per condition. ( I ) n = 4 mice per condition. Bars in graphs show mean values, circles show individual data points, and error bars show SDs. The P values between data sets are from unpaired 2-tailed t tests. Scale bars: 400 um ( A ) or 100 uM ( D and G ).

Supportive validation

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Experimental details: Figure 2 Hyperoxia reduces postnatal proliferation and survival of left atrial cardiomyocytes in neonatal mice. ( A and B ) Sections of left atrial appendages from P4 control ( A ) and hyperoxia-exposed ( B ) mice stained with phosphorylated Histone H3 (pHH3, green), cardiac troponin T (TNNT2, red), and DAPI (blue). Arrows point to pHH3 + cardiomyocytes. ( C ) Percentage of pHH3 + cardiomyocytes in P4 mice exposed to room air or hyperoxia. n = 4 mice per condition. ( D and E ) Sectioned left atrial appendages of P4 control ( D ) and hyperoxia-exposed ( E ) mice stained for the active, cleaved form of Caspase 3 (cl-Casp3), TNNT2, and DAPI. Arrows point to cl-Casp3 + cardiomyocytes. ( F ) Percentage of cl-Casp3-labeled CMs in the left atria of P4 hyperoxia-exposed and control mice. Room air, n = 4; hyperoxia, n = 5 mice per condition. Box plots show median, second quartiles, and third quartiles; whiskers indicate range, and circles represent individual data points. P values are results of unpaired 2-tailed t tests. Scale bars: 200 mum.

Supportive validation

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Experimental details: Figure 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 mum. ( 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.

Supportive validation

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Experimental details: Figure 8 Hyperoxia suppresses fatty acid synthesis genes in left atrial tissue explanted from human infants. ( A ) Results of qPCR for Fasn and Scd1 in left atrial tissue explanted from human infants who died at birth due to anencephaly and exposed to room air or hyperoxia for 24 hours. n = 5 donors. ( B ) Sectioned explants exposed to room air (top) or hyperoxia (bottom) were stained for SCD1 (green), TNNT2 (red), and DAPI (blue). ( C ) Graph shows staining intensities for SCD1 in sections of control and hyperoxia-exposed mice determined using NIH ImageJ 2.0/Fiji. n = 4 donors. ( D ) Sections of explants exposed to room air (top) and hyperoxia (bottom) stained for the proliferation marker Ki67 (green), TNNT2 (red), and DAPI (blue). ( E ) Graph shows percentages of TNNT2-expressing cells with Ki67 + nuclei in explants exposed to room air or hyperoxia. n = 7 donors. ( A , C , and E ) Circles indicate individual values for explants of each donor. Boxes show medians and inner quartiles; whiskers represent the range. P values are the results of either single-sample 1-tailed t test and Wilcox test ( A ) or unpaired 2-tailed t tests ( C and E ). ( B and D ) Scale bars = 100 mum.

Supportive validation

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Experimental details: Figure EV2 Cardiomyocyte quantification and characterization by FACS and qRT-PCR FACS staining for cTNNT2 in D10 and D15 human ES cell-derived cardiac cells. IgG 1 isotype was used as a control. POU5F1 mRNA expression levels in human ES cells and D15 as well as D30 cardiac cells, as measured by qRT-PCR. Expression levels were normalized to a non-oscillatory housekeeping gene ( PPIA ). Data are represented as mean +- s.e.m. of three independent replicates.

Supportive validation

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Experimental details: Figure 2 Characterization of AAV-hTCF21-GFP and AAV-hTCF21-FLAG-YAP expression in mouse hearts. Wild type C57BL/6J mice were administered AAV-hTCF21-GFP, AAV-hTCF21-FLAG-YAP, or no virus control. Hearts were excised and processed for immunostaining or cell isolation and fractionation. ( a ) Heart sections were stained to detect GFP or FLAG (green), and counterstained to visualize Hsp47, alphaSMA, CD31, or cardiac troponin T (red) and nuclei (DAPI). Right column panels are higher magnification merged images. Scale bar, 100 mum. ( b-e ) Heart cells were isolated from mice administered AAV-hTCF21-GFP or AAV-hTCF21-FLAG-YAP and separated into cardiomyocyte-enriched (CM) and non-myocyte-enriched (NM) fractions. RNA and protein were isolated for qPCR analysis and immunoblotting, respectively. ( b-d ) Expression of GFP, FLAG-Yap, and cardiac troponin T (Tnnt2) mRNA was determined by qPCR. ( e ) Protein fractions (20 mug per sample) were analyzed by western blot to detect YAP, PDGFRalpha, Hsp47, and cTnT protein levels. Whole liver homogenates from AAV-hTCF21-GFP and AAV-hTCF21-FLAG-YAP transduced mice (G and Y, respectively) were also analyzed (5 mug per sample). N = 3 mice/group. Representative blots are shown. *p < 0.05. Full-length blots are presented in Supplementary Fig. S3 .

Supportive validation

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Experimental details: Figure 6 Transduction with AAV-hTCF21-FLAG-YAP upregulates inflammatory markers in the heart. Wild type C57BL/6J mice were administered AAV-hTCF21-GFP or AAV-hTCF21-FLAG-YAP. ( a-c ) RNA was isolated from LV tissue for qPCR analysis. ( d , e ) Hearts were sectioned for immunostaining to detect CD68 + macrophages (green), cardiac troponin T (red), and nuclei (DAPI). White arrows identify CD68-positive cells. Quantification of CD68-positive cells in panel ( e ). Scale bar, 100 mum. ( f ) IL-1b mRNA was measured in LV tissue by qPCR. N = 3 mice/group. *p < 0.05. ns not significant.

Supportive validation

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Experimental details: Fig. 1 HFOs recapitulate patterns of early cardiomyogenesis. a , The protocol for HFO formation. hPSC aggregates were individually embedded in Matrigel and differentiated using CHIR and IWP2. The figure shows the development of HFOs from d-4 until d10 of differentiation (upper row) as well as MIXL1-GFP and NKX2.5-eGFP expression patterns (bottom row). b , A typical HES3 NKX2.5-eGFP-derived HFO forming three layers: IC, ML and OL. c , An HFO rotated around its virtual axis and respective schemes. d , Whole-mount IF staining for the myocardial marker MHC. e , A paraffin section stained for cTnT and an anti-GFP antibody. f , A scheme of an HFO with highlighted cardiomyocytes. g , Sequential paraffin sections stained for cTnT and the ST marker WT1. h , A scheme of an HFO with highlighted ST-like cells. i , Whole-mount IF staining for the mesenchymal cell marker vimentin. j , A scheme of an HFO with highlighted mesenchymal cells. Scale bars, 500 um ( a ) and 200 um ( b - e , g , i ).

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Experimental details: Fig. 3 Differential expression of SOX17, SOX2 and HNF4alpha reveals formation of distinct foregut endoderm tissues. a , H&E staining (left column) and staining for the endoderm marker SOX17 (right column) with enlargements showing endodermal cavities (END CAV) in the IC and endodermal islands (END ISL) in the OL. b , TEM images of endodermal cavities in the IC with microvilli (arrows). c , d , Cryosections of HFOs stained for cTnT and the AFE marker SOX2 ( c ) or the hepatocyte marker HNF4alpha ( d ). The arrows point at endodermal islands in the OL. e , A scheme of an HFO with highlighted distinct endoderm tissues. Scale bars, 100 um ( a ), 5 um ( b ) and 200 um ( c , d ).

Supportive validation

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Experimental details: Fig. 1 ACE2 expression in adult human tissues and iPS-derived cells. (A) ACE2 expression in adult human tissues based on Protein Atlas and Allen Brain Atlas databases. Consensus normalized expression (NX) and z-score are used to represent the relative abundances within the respective databases (Citations in the main text). NA = not analyzed in the database. Characterization of NP and CM. NP were cultivated for 7 days and CM were differentiated for 35 days and characterized by IF (B-D and F) and flow cytometry (E). NP were immunostained for (B) Nestin (neural progenitors), (C) S100b, (astrocytes); and (D) MAP-2 (neurons). (E) Representative flow cytometry graph showing a quantification of 83.2% TNNT2 + cells from one of the cell batches used in this study; (F) CM immunostained for cardiac troponin T (TNNT2). Scale bars: (B-D) 40 um and (F) 250 um. (G) Relative mRNA expression levels of ACE2 in iPSC-derived cultures; NP cultivated for 7 days and CM differentiated for 35 days. (n = 3 cell culture batches for both), normalized to reference genes, GAPDH and HPRT-1. The data correspond to 3 independent cell lines for NP.

Supportive validation

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Experimental details: Figure 1 cP1P enhances cardiac differentiation in human embryonic stem cells. ( A ) Illustration of the chemically defined cardiac differentiation protocol utilized in this study. cP1P was administered during different stages of cardiac differentiation. Group 1 represents treatment with cP1P from days -5 to 0; Group 2, from days 5 to 8; and Group 3, from days 0 to 8 during differentiation. ( B ) Immunoblot analysis of the cardiomyocyte marker TNNT2 in Groups 1, 2, and 3 were analyzed at day 8 of cardiac differentiation. Graph represents the relative protein expression of TNNT2 as normalized to GAPDH ( n = 3, ** p < 0.01, *** p < 0.001). ( C ) Immunofluorescence analysis of the cardiomyocyte marker TNNT2 in Groups 1, 2, and 3 were analyzed at day 8 of cardiac differentiation, Scale bar = 10 mum. ( D ) Gene expression analysis of cP1P-treated cardiomyocytes by qRT-PCR at day 7 of cardiac differentiation showed higher expression levels of NKX2.5 , MEF2C , TBX5 , GATA4 , and TNNT2 in Group 3 when compared to their respective controls. Data are presented as the means +- SDs of three independent experiments ( n = 3, * p < 0.05, ** p < 0.01).

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Experimental details: Figure 2 Bioactive lipid cP1P augments beating colony number and contracting area by activating SMAD signaling in human embryonic stem cells (hESC)-derived cardiomyocytes. ( A ) Representative image of the number of beating cardiomyocytes in the control (DMSO-treated) and cP1P-treated groups at day 6. Scale bar = 500 mum. ( B ) Cell number measurements of beating cardiomyocytes. hESCs were seeded into 12-well plates (7.5 x 10 4 cells/well), treated with DMSO (control) or cP1P as indicated, and induced to differentiate for 8 days. Data are presented as the means +- SDs of three independent experiments ( n = 3, * p < 0.05). ( C ) Areas of contraction of the beating clusters obtained from the control or cP1P groups at day 8 were calculated by ImageJ software using the captured images. Contracting areas were individually marked using ImageJ tools and a calibration of 0.46 pixels/mum was used to measure the area. Data are presented as the means +- SDs ( n = 11, ** p < 0.01). ( D ) Beating rates for the control and cP1P groups were measured as beats/min. Data are presented as the means +- SDs ( n = 3, ns = non-significant). ( E ) The expression levels of p-ERK, p-SMAD 1/5/8, total ERK, and SMAD 1/5/8 were analyzed along with the expression levels of cardiogenic transcriptional factors NKX2.5, TNNT2, and MLC2V by Western blot during CM differentiation in DMSO-treated control and cP1P-treated CMs at the indicated time points.

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Experimental details: Figure 3 cP1P promotes cardiac differentiation in hESCs by partially regulating S1PR-mediated SMAD1/5/8 signaling. ( A ) The activity of RHOA/ROCK1/SMAD signaling with or without cP1P was tested by evaluating the expression of RHOA and ROCK1 by Western blot at the indicated time points along with GAPDH expression as a reference. ( B ) Schematic representation of the treatment with S1PR inhibitor VPC during days 3 to 5 of CM differentiation coinciding with IWR1 inhibitor treatment. ( C ) The activity of RHOA/ROCK1/SMAD signaling was inhibited by the administration of VPC, as shown by the downregulation of RHOA, ROCK1, and p-SMAD 1/5/8, but cP1P treatment could partly rescue the inhibitory effect of VPC, as shown by the rescue of p-SMAD 1/5/8, demonstrated by Western blot. The effects of S1PR inhibition by VPC on cardiomyocyte differentiation, shown by ( D ) the downregulation of NKX2.5 at day 5. ( E ) Effect of VPC treatment between days 3 to 5 on the expression of TNNT2 and MLC2V at day 8 of CM differentiation showed that cP1P treatment could partially rescue the inhibitory effects of VPC treatment, as demonstrated by Western blot. ( F ) Schematic representation of the treatment with VPC during days 5 to 8 of CM differentiation. ( G ) Immunoblots of RHOA, ROCK1, p-SMAD1/5/8, SMAD1/5/8, and NKX2.5 at day 5 of VPC treatment in DMSO-treated controls and cP1P-treated CMs. ( H ) Immunoblots of TNNT2 and MLC2V at day 8 of VPC treatment in DMSO-treated controls and cP1P-treated CMs.

Supportive validation

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Experimental details: Figure 4 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.

Supportive validation

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Experimental details: FIGURE 1 Cardiac differentiation from hiPSCs (A) Schematic of the protocol for the differentiation of cardiomyocytes from hiPS cells with (RA (+)) or without RA (RA (-)) treatment (B) The ratio of cardiomyocytes quantified by immunostaining with cardiac troponin-T (cTnT). The proportion of cTnT positive cells was 93.91 +- 2.51% in CT (control hiPS-CMs), or 96.79 +- 1.81% in AM (RA-treated hiPS-CMs), respectively (P = N.S.). Error bars represent SD of the mean from the values of independent experiments. n = 3. N.S., not significant (C) Immunofluorescence analysis of the percentage of MLC2a (atrial isoform) and MLC2v (ventricular isoform) positive expression in the CT and AM. Error bars represent SD of the mean from the values of independent experiments. ** p < 0.01 for comparison with MLC 2a+/MLC 2v-cardiomyocyte in CT, ## p < 0.01 for comparison with MLC 2a+/MLC 2v-cardiomyocyte in AM, n = 3.

Supportive validation

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Experimental details: Fig. 3 Gastrin decreases cardiomyocyte apoptosis and fibrosis after myocardial infarction. a Representative images ( a1 ) and quantification ( a2 ) of the fibrotic area in infarct border zone by Masson trichrome staining at 28 days post-MI. b Representative images ( b1 ) and quantification ( b2 ) of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive (TUNEL + ) cardiomyocytes in infarct border zone at 2 days after MI. TUNEL + nuclei are stained green, cardiac troponin T antibody (cTnT) is red, and 4'-6-diamidino-2-phenylindole (DAPI) is blue. b3 Western blot was used to measure the protein levels of Caspase 3 in the infarct border zone at 2 days after MI. Results are expressed as the ratio of cleaved caspased 3 to total caspase 3. c , Representative images ( c1 ) and quantification ( c2 ) of cardiomyocyte proliferation 28 days after myocardial infarction, through Ki67 immunostaining. Cardiac troponin T antibody (cTnT) is green, Ki67 is red, and 4'-6-diamidino-2-phenylindole (DAPI) is blue. a and c Scale bar = 50 um. b Scale bar = 20 um. n = 8. * P < 0.05, vs. MI mice. P = NS means no significant difference

Supportive validation

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Experimental details: Figure 7 In vitro cardiac differentiation of TMRM-low and high cells. Expression of early cardiac gene/protein: T-Box transcription factor 5 ( TBX5 ) and late cardiac genes cTNT ( TNNT2 ) and alpha-sarcomeric actin ( ACTC1 ) in TMRM-low and high cells after 1 week of culture in cardiomyogenic differentiation media. The expression of these genes was analyzed by qRT-PCR ( n = 5) ( A ), and Western blot ( n = 4) ( B ) Data are represented as mean +- SD of the fold change. Statistical differences were calculated significant as * p < 0.05, determined by paired Student's t -test.

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Experimental details: Figure 1 Serum progesterone concentration declines after birth and is associated with decrease in CM proliferation. A,B, Evaluation of CM proliferation by Ki67 and PH3 immunostaining at E16, P1, P7 and P14 hearts. Representative images with z-stacking are shown in A1 and B1, and quantification of percentages of Ki67 + and PH3 + CMs is shown in A2 and B2, respectively (n = 5). Scale bars are 20 um. cTnT indicates cardiac troponin T. C, Serum concentrations of progesterone in mice at E16, P1, P7 and P14 detected using ELISA kit (n = 5-8). *** P < .001 vs E16; ### P < .001 vs P1; &&& P < .001 vs P7. CM, cardiomyocyte

Supportive validation

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Experimental details: Figure 7 Knock-out of Setd4 inhibits cardiomyocyte apoptosis in MI-injured mice. ( A , B ) Representative immunofluorescence ( A ) and quantification ( B ) for recombinant cells. Scale bars = 500 mum. In the three areas of the MI-injured left ventricle after four weeks of Setd4 knock-out. Scale bars = 500 mum. ( C , D ) Immunostaining ( C ) and quantification ( D ) for recombinant cells of capillary (Td + FABP4 + ) in the three areas of the MI-injured left ventricle. Scale bars = 50 mum. ( E , F ) Immunostaining for alpha-SMA (Scale bars = 200 mum) ( E ) and Troponin T (Scale bars = 50 mum) ( F ) after four weeks of Setd4 knock-out. ( G ) Representative immunofluorescence and quantification for apoptotic cardiomyocytes by performing TUNEL assay after two weeks of Setd4 knock-out. Scale bars = 50 mum. Nuclei were stained with DAPI. All data are represented as mean +- SEM. n = 4 mice. Unpaired t test for ( F , G ), two-way ANOVA with Bonferroi's correction for multiple comparisons test for ( B , D ). ** p < 0.01. ns not significant, IR infarction region, BZ border zone, NIR non-infarction region.

Supportive validation

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Experimental details: Fig. 2 Nrf1 deletion impairs neonatal heart regeneration. a Illustration showing timeline of MI and Sham surgeries performed at P3 and subsequent days of sample collection. b , c Fractional shortening ( b ) and ejection fraction ( c ) of Nrf1 fl/fl and Nrf1 cKO mouse hearts at 25 days after MI or Sham surgery; n = 4 animals for Sham groups and n = 7-8 animals for MI groups; ** p = 0.0022 ( b ), ** p = 0.0009 ( c ) by Student's t -test two-tailed. d Masson's trichrome staining showing fibrotic scarring in Nrf1 fl/fl and Nrf1 cKO mouse hearts at 25-day after MI; similar results were observed in six animals; Scale bar, 200 mum. e Immunohistochemistry showing pH3 + cardiomyocytes (cTnT + ), marked by arrows, in heart sections collected from Nrf1 cKO and Nrf1 fl/fl mice 3-day post-MI; Scale bar, 50 mum. f Quantification of pH3 + cardiomyocytes (cTnT + ) in heart sections from ( e ); n = 12 animals for WT and n = 11 animals for Nrf1 cKO, ** p = 0.0034 by Student's t -test two-tailed. g Chymotrypsin-like activity of the proteasome in hearts from Nrf1 cKO and Nrf1 fl/fl mice at P4. arb. units, arbitrary units; n = 6 animals for each group; * p = 0.0373 by Student's t -test one-tailed. h UMAP visualization of cardiomyocyte clusters colored by identity. CM1 and CM3 were merged as a single cluster due to their transcriptome similarity. i Fraction of cardiomyocyte populations in Nrf1 fl/fl and Nrf1 cKO hearts in MI or Sham conditions; n = 4-6 animals for each group examined over 1 indepe

Supportive validation

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Experimental details: Figure 6 Transplanted hESC-CM grafts interact with a diffuse Purkinje conduction system in the porcine myocardium An hPSC-cardiomyocyte graft 2 weeks post-transplantation, marked by human-specific slow skeletal cardiac troponin I (ssTnI) (red) (), interacts with Cx40-positive (white) Purkinje fibers. Wheat germ agglutinin (WGA) (green) delineates cardiac sarcolemma. Purkinje-transitional cell-graft (left panel) and direct Purkinje-graft (right panel) interactions are observed. Sale bars, 100 or 20 mum (magnified).

Supportive validation

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Experimental details: Figure 1 Co-culture of cells plated at an initial high cell density enhanced the cardiomyocyte differentiation efficiency of cells plated at an initial low cell density. ( a ) Schematic of the previously established protocol of cardiomyocyte differentiation . ( b ) Schematic of co-culture experiments. The cells were plated as follows. Cells were plated at a low cell density of 5 x 10 3 cells/cm 2 and high cell density of 2 x 10 5 cells/cm 2 . Condition 1: Only low cell density cells in the lower layer (L indicates lower). Condition 2: Only high-density cells in the upper layer (U indicates upper). Conditions 1 and 2 were used as controls. Condition 3: Co-culture of high-density cells in the upper layer and low cell density cells in the lower layer to assess cell-secreted signals. Condition 4: The same total number of cells at an initial low and high density, as specified in condition 3, was plated on the same surface to assess both cell-secreted signals and cell-cell contact signals. ( c ) The percentage of cTnT-positive cells measured by flow cytometry on day 14. Mean +- standard error (SE), * P < 0.05, t-tests with Holm's correction, ns: non-significance, n = 3.

Supportive validation

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Experimental details: Figure 3 Higher concentrations of Wnt inhibitor did not increase cardiac gene expression at an initial low cell density. ( a ) Expression of anti-cardiac and cardiac progenitor genes on day 5, treated with 5 and 10 muM IWP2 during days 3-5. ( b ) Flow cytometry for cTnT on day 14, in cells treated with 5 and 10 muM IWP2 during day 3-5.

Supportive validation

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Experimental details: Figure 4 Early addition of Wnt inhibitor during days 1-3 was sufficient to improve cardiomyocyte differentiation efficiency at a low initial cell density. ( a ) Schematic of the new protocol of cardiomyocyte differentiation at an initial low cell density, with early addition of IWP2 from day 1-3. ( b ) Expression of anti-cardiac mesoderm genes CDX2 and MSX1 and cardiac progenitor genes ISL1 and NKX2.5 on day 5. The expression levels of cardiomyocyte genes ( MYL2 , TNNI3 , and TNNT2 ) on day 14. IWP2 was added during days 1-3 or days 3-5. ( c ) Flow cytometry of cTnT-positive cells induced by IWP2 treatment during days 1-3 and days 3-5. ( d ) The percentage of cTnT-positive cells under the same conditions as in ( c ). Mean +- SE, ** P < 0.01, t-tests with Dunnett's correction, n = 4.

Supportive validation

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Experimental details: Figure 1. Identification of activators and inhibitors of 5F-mediated cardiac reprogramming from a human ORF cDNA screen. ( A ) Schematic diagram of the human ORF cDNA library screen strategy for cardiac reprogramming in adult TTFs. ( B ) Venn diagram showing the number of activators identified from the screen. Genes with Z -scores of alphaMHC-GFP or cTnT expression >=2 were defined as activators. Twenty-five genes induced alphaMHC-GFP expression only, 35 genes induced cTnT expression only, and 11 genes induced expression of both markers. ( C ) Venn diagram showing the number of inhibitors identified from the screen. Genes with Z -scores of alphaMHC-GFP or cTnT expression -2 or lower were defined as inhibitors. One-hundred-twenty-one genes repressed alphaMHC expression only, 41 genes repressed cTnT expression only, and 33 genes repressed both alphaMHC-GFP and cTnT expression. ( D ) Representative immunocytochemistry images of TTFs from adult alphaMHC-GFP transgenic mice treated with 5F and either empty virus or viruses encoding activators (ZNF281 or PHF7) and inhibitors (FOXA3 or SOX9). Cells were fixed and stained for alphaMHC-GFP (green), cTnT (red), and Hoechst (blue) 9 d after infection. Bars, 2 mm. ( E ) Pathways enriched in activators ( n = 49) and inhibitors ( n = 129), respectively, by DAVID pathway analysis.

Supportive validation

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Experimental details: Figure 2. ZNF281 enhances cardiac reprogramming of adult fibroblasts. ( A ) Immunocytochemistry images of adult alphaMHC-GFP transgenic TTFs 7 d after infection with empty, ZNF281, 5F, or 6F retroviruses show that ZNF281 enhances expression of cardiac markers with 5F. (Green) alphaMHC-GFP; (red) cTnT; (blue) Hoechst. Bars, 500 um. ( B , C ) Representative flow cytometry plot ( B ) and analyses ( C ) of alphaMHC-GFP + and cTnT + TTFs 7 d after infection with empty, 5F, or 6F. ( D ) TTFs were infected with empty, 5F plus empty, and ZNF281 for 7 d. Transcript levels of cardiac marker genes ( Myh6 and Actc1 ) and fibroblast marker genes ( Col1a2 and Sox9 ) were determined by qPCR. (*) P < 0.05.

Supportive validation

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Experimental details: Figure 6. ZNF281 represses the inflammatory response through the NuRD complex. ( A ) TTFs were infected with empty, 5F plus empty, ZNF281, or each individual NuRD complex subunit retrovirus for 7 d. Transcript levels of inflammatory ( IL6 and Ccl2 ) and cardiac ( Myh6 and Actc1 ) marker genes were determined by qPCR. ( B ) Representative flow cytometry plot of alphaMHC-GFP + and cTnT + TTFs 7 d after infection with empty, 5F plus empty, ZNF281, or each individual NuRD complex subunit retroviruses. The dashed line indicates control. (*) P < 0.05.

Supportive validation

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Experimental details: Figure 7. Anti-inflammatory drugs promote cardiac reprogramming. ( A ) 5F reprogrammed TTFs treated with either DMSO or the indicated anti-inflammatory drugs for 7 d after infection. Expression of inflammatory genes ( IL6 , Ccl2 , and Ptgs1 ), and cardiac genes ( Myh6 , Actc1 , and Nppa ) was determined by qPCR. (Dex) 10 uM Dex; (Nab) 10 uM Nab. ( B ) Immunocytochemistry images of 5F reprogrammed adult alphaMHC-GFP transgenic TTFs treated with DMSO or the indicated anti-inflammatory drugs for 7 d. (Green) alphaMHC-GFP; (red) cTnT; (blue) Hoechst. Bars, 500 um. ( C , D ) Representative flow cytometry plot ( C ) and analyses ( D ) of alphaMHC-GFP + and cTnT + cells in 5F reprogrammed adult alphaMHC-GFP transgenic TTFs treated with DMSO or the indicated anti-inflammatory drugs for 7 d. (*) P < 0.05. ( E ) Model showing the mechanism of action of ZNF281 on 5F-mediated direct cardiac reprogramming. ZNF281 is a cardiac transcription coactivator recruited by GATA4 to cardiac enhancers to activate cardiac gene expression. ZNF281 also represses the inflammatory response, which acts as a barrier pathway to cardiac reprogramming.

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Experimental details: Figure 1 ( A ) Schematic representation of direct cardiac reprogramming protocol. MEFs or CFs were plated on Matrigel pre-coated plates in fibroblast growth medium containing 1 muM PTC-209 (PTC) or DMSO (NT) one day before the addition of Cardiac Reprogramming Medium (CRM) containing the small molecule cocktails. At day 16 for MEFs or 20 for CFs, the medium was changed into Cardiomyocyte Maintaining Medium (CMM) for at list 10 days more. Samples were analysed at final stage of reprogramming (day 24 or 28, for MEFs and CFs respectively). ( B,C ) Representative 2D scatter plots showing end-stage reprogrammed cell population upon 24 h PTC-209 pre-treatment (PTC) or DMSO (NT). Green dots represent alpha-MHC + cells. Typical results obtained starting from MEFs are in ( B ); those starting from CFs are in ( C ). ( D,E ) Representative plots showing fluorescent cells vs total cell counts, with the percentage of alpha-MHC + cells highlighted in green, at final stage of reprogramming. The increasing in alpha-MHC + cells upon PTC-209 pre-treatment is shown in ( D ) for MEFs and in ( E ) for CFs. ( F,G ) Box plots showing the percentage of alpha-MHC + cells, normalized to cell count, at final stage of reprogramming from MEFs ( F ) or CFs ( G ). For each data set: n = 4; Delta = mean of % of alpha-MHC + cell increase upon 24 h PTC-209 pre-treatment. *p < 0.05, **p < 0.01. ( H ) Representative immunostaining of cardiac marker alpha-MHC (green). ( I ) Representative immunostaining of cardi

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Experimental details: Figure 7. Heterogeneity and Distinct Injury Response of Immune Cells (A) UMAP representation of different myeloid cell populations analyzed. (B) Heatmap showing expression of top enriched genes for each myeloid cell sub-population. (C-G) UMAP plots showing expression of Cd86 , an M1-macrophage marker gene (C); Arg1 , an M2-macrophage marker gene (D); Plac8 , a monocyte-enriched gene (E); Ly6c2 , an M1 monocyte-enriched gene (F); and Naaa , a DC-like cell enriched gene (G). (H) Percentage of each immune cell population over total nonmyocytes within each sample. (I) UMAP plot showing expression of Clcf1 among different cell types present in the P1+1 dpi and dps scRNA-seq data. (J) Representative images showing EdU incorporation (magenta) and cardiac troponin T (cTnT) immunofluorescent staining (green) of NRVM cells treated with 200 ng/mL BSA (negative control), 20 ng/mL recombinant insulin-like growth factor 2 (IGF2) (positive control), or different concentrations of recombinant CLCF1 (5, 10, 25, and 50 ng/mL). Scale bar, 100 mum. (K) Quantification showing the proportion of EdU-positive cells among cTnT-positive cells (CM) after treatment of BSA, CLCF1 recombinant protein, or recombinant IGF2 (n = 4 per each group; **p < 0.01, ****p < 0.0001). See also Figure S7 .

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Experimental details: FIGURE 3 Cardiomyocyte-like cells induced by GMTMS from MICFs express multiple structural proteins. (A) Immunostaining for cTnT, cTnI, and alpha-Actinin at day 21 after GMTMS transduction in MICFs derived from Myh6 -mCherry mice. mCherry + cells expressed cardiac proteins with well-organized sarcomeric structures. (B) Representative images of GMTMS-induced iCMs co-expressing cTnT and cTnI. (C) Immunofluorescence for cTnT (red) and cTnI (green) was performed 3 weeks after transduction of MICFs (from C57 mice, 8 weeks old) with GMT, GMTM, GMTS, GMTMS, and GMTMM. (D) Quantification of cTnT and cTnI double positive cells ( n = 3). All data are presented as mean +- SD. ** p < 0.01 vs. relevant control. Scale bars: 100 mum.

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Experimental details: FIGURE 6 GMTMS significantly increase the iCMs reprogramming efficiency from myofibroblasts traced by Postn . (A,B) Immunofluorescence for cTnI and cTnT was performed 3 weeks after transduction of indicated cocktails with MICFs (from Postn -MCM +/- ; Rosa26 -lsl-tdTomato +/- mice, 8 weeks old). (C) Quantification of cTnI- or cTnT- positive iCMs induced from Postn -tdTomato + myofibroblast by GMTMS and GMT ( n = 5). (D) Quantification of cTnI- or cTnT- positive iCMs induced by GMTMS from Postn -tdTomato + myofibroblast or Postn -tdTomato - MICFs ( n = 5). All data are presented as mean +- SD. ** p < 0.01 vs. relevant control and ns means no significant difference. Scale bars: 100 mum.

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Experimental details: FIGURE 7 Myocd or Sall4 significantly enhance iCMs reprogramming efficiency from adult cardiac fibroblasts. (A) Representative images of GMTMS-induced iCMs co-expressing cTnT and cTnI. (B) Immunofluorescence for cTnT (red) and cTnI (green) was performed 3 weeks after transduction of ACFs (from C57 mice without injury, 8 weeks old) with GMT, GMTM, GMTS, GMTMS, and GMTMM. (C) Quantification of cTnT and cTnI double positive cells ( n = 3). Note that four factors (GMTM or GMTS) are enough to reprogram abundant iCMs co-expressing cTnT and cTnI. ** p < 0.01 vs. relevant control and ns means no significant difference. Scale bars: 100 mum.

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Experimental details: Figure 1. Blood netrin-1 increases in MI patients and mice with myocardial IR surgery. (A) Blood netrin-1 levels of healthy donors and MI patients ( n = 10 for healthy control, n = 9 for MI; two-tailed Welch's t test). (B) Experimental strategy for the murine myocardial IR model. (C) Infarct sizes of IR group compared with the sham group ( n = 4 per group; two-tailed unpaired t test). (D) Representative infarct staining results for the IR model. The infarct area is outlined by a green line; the blue line marks the AAR (scale bar = 1 mm). (E) cTnI levels after surgery ( n = 6 for sham group, n = 9 for IR group; two-tailed Welch's t test). (F) Murine blood netrin-1 levels were measured in sham, ischemia (45 min), IR2h (45 min of ischemia + 2 h of reperfusion), and IR4h (45 min of ischemia + 4 h of reperfusion) groups ( n = 6 for sham group, n = 5 for ischemia group, n = 9 for IR2h group, and n = 5 for IR4h group; one-way ANOVA with Bonferroni post hoc tests). (G) Immunofluorescence costaining of netrin-1 with cTnT and Ly6G in WT mice after sham or IR surgery shows netrin-1 in cardiomyocytes and neutrophils. (For single-channel pictures, scale bar = 2 um; for merged channel pictures, scale bar = 8 um.) *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data are presented as mean +- SD.

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Experimental details: Figure 6 ODNcap-F protects pulmonary vein cardiomyocytes from allergic injury. Lung total RNA and sections were prepared on Day 70. (A) Expression levels of cardiac muscle-specific genes in the lung were measured by real-time quantitative PCR ( n = 12). Data are shown as Tukey's box plots. *** P < 0.001; n.s., not significant by Kruskal-Wallis test with Dunn's multiple comparisons. (B) Lung sections prepared on Day 70 were stained with anti-cardiac muscle troponin T (cTnT) antibody ( n = 6). Representative images are shown. Positive reaction of the antibody (red) was observed around the large pulmonary veins. Injured areas (arrows) of the cTnT-positive cells were observed in Ctrl-F and Cap-F, but not in NT or ODNcap-F. Scale bars, 100 mum.

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Experimental details: 5 FIGURE Local proliferation of noncardiomyocytes contributes to fibrosis. (a) Co-staining of cTnT+ cardiomyocytes and pH 3+ cells reveals an increase in nonmyocyte proliferation at 14 DPI specifically in the injured zone (note: DAPI omitted in larger panels; insets show higher magnification views within the same region of the heart, but are not enlargements of the larger panels). Quantification of proliferating cell types reveals that (b) cardiomyocytes do not proliferate to repopulate the injured region at any point after injury (proliferative cardiomyocytes quantified as those positive for both pH 3 and cTnT; Injured Region: n = 4 for 1, 4, 7, and 14 DPI; n = 5 for 28 and 56 DPI; n = 6 for uninjured; Remote Region: n = 4 for 1, 4, and 7 DPI; n = 5 for 14, 28, and 56 DPI; n = 6 for uninjured), but (c) a proliferating nonmyocyte population within the injured zone is present at 14 and 28 DPI (Injured Region: n = 3 for 1 and 4 DPI; n = 4 for 7 and 14 DPI; n = 5 for 28 and 56 DPI; n = 6 for uninjured; Remote Region: n = 4 for 1, 4, and 7 DPI; n = 5 for 14, 28, and 56 DPI; n = 6 for uninjured; large scale bar = 100 mum, small scale bar = 5 mum; ** p

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Experimental details: Morphological analysis of EHTs in four different media at day 21 of culture. ( A ) Brightfield images of full EHTs in BPEL+HS, DMEM+HS, MM+HS, or MM(SF). Scale bar = 0.5 mm. ( B ) Confocal images of whole-mount tissue immunostaining for cardiac troponin T (cTnT, red) counterstained with DAPI (nuclei, blue). Scale bars, 25 um. HS, horse serum and SF, serum-free.

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Experimental details: Figure 5 aCD47 reduces the expression of Bax and p-p38 MAPK in Dox-treated cardiomyocytes. (A) Immunostaining for the expression of cTnT, cleaved caspase-1/3, Bax and p-p38 MAPK in the treated cardiomyocytes. Cardiomyocytes were identified as cTnT-positive cells (green). Red, cells positively stained for cleaved caspase-1/3, Bax and p-p38 MAPK. Representative images with x200 magnification. (B) Semi-quantitative analysis of positively stained cells by ImageJ software. Data are presented as the relative intensity of positively stained cells over untreated controls. (C) Western blot analysis for the expression of pro-caspase-1, cleaved caspase-1, cleaved caspase-3, Bax and Bcl-2 in the treated cardiomyocytes. GAPDH was internal loading control. One representative blot. (D) p-p38 MAPK + cells after Dox and aCD47 treatment were analyzed by flow cytometry. Data are presented as the percentage of p-p38 MAPK + cells. (E) Association between the percentage of p-p38 MAPK + cells and apoptotic cardiomyocytes after treatment. Bar plot data in all panels are represented as mean +- standard error. n=3. * P

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Experimental details: FIGURE 3 Post-MI myocardial upregulation of UCHL1 occurred primarily in the cardiomyocytes of the peri-infarct zone in humans. Paraffin-embedded and formalin-fixed ventricular myocardial sections (4-mum thick) from human non-failing donor hearts (NFH) or from the tissue cylinder core obtained from acute myocardial infarction (AMI) patients during the installation of a LV assisting device were subjected to deparaffinization, rehydration, antigen recovery, and indirect immunofluorescence staining for cardiac troponin T (cTnT) to identify cardiomyocytes (green) and for UCHL1 (red) as described in the Methods section. Nuclei are counter-stained with DAPI (blue). Shown are the representative confocal images. Scale bar, 100 mum.

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Experimental details: Exposure to SMG does not cause cytoskeletal deteriorations in CMs (A) A representative immunofluorescence staining of sarcomeric alpha-actinin (ACTN2) and cardiac troponin T (cTnT) in c-CMs and SMG-CMs (scale bar: 10 mum). (B) TEM images of SMG-CMs and c-CMs. Black arrow heads indicate caveolae structures (black arrows show stress fibers (scale bar: 500 nm). (C) Western blot analysis of Caveolin (CAV1) in control and SMG-exposed CMs (molecular weight of 22 kDa). GAPDH (36 kDa) was used as an internal protein loading control.

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Experimental details: FIGURE 7 Results of cTnT fluorescence microscopy images of the iPSC-CM differentiation experiment. TE stands for the traditional enhancement method, CIEGAN, CIEGAN plus (CIEGANP) is our method, GT is the ground truth, and EGT is the enhanced ground truth.

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Experimental details: FIGURE 3. TRPV1 activation protected against PE-induced cardiomyocyte hypertrophy in vitro. A, Representative images and analysis of the cell surface area of neonatal rat cardiomyocytes stained with cTnT (green) and DAPI (blue) (n = 5, scale bar = 10 mum). B, The ANP, BNP, beta-MHC protein expression in primary cardiomyocytes treated with capsaicin and PE (n = 5). C, Quantitative polymerase chain reaction analyses of the mRNA levels of ANP, BNP, beta-MHC in the indicated groups. n = 5 per group. * P < 0.05 compared with control, # P < 0.05 compared with PE group.

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Experimental details: FIGURE 6. Disruption of MAM by siMFN2 abolished capsaicin-mediated mitochondria protection. A, MMP was assessed using a JC-1 kit. The JC-1 aggregate/JC-1 monomer ratio was measured for MMP (n = 6, scale bar = 10 mum). B, Effects of capsaicin on ROS production in PE-treated cardiomyocytes using MitoSOX red staining (n = 5, scale bar = 10 mum). C, ATP levels in cultured cardiomyocytes (n = 6). D, Representative images and analysis of the cell surface area of neonatal rat cardiomyocytes stained with cTnT (green) and DAPI (blue) (n = 5, scale bar = 10 mum). * P < 0.05 compared with control, # P < 0.05 compared with PE group, $ P < 0.05 compared with PE + CAP group.

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Experimental details: Fig. 8 TBX5-Lineage tracing confirms devCellPy prediction of a predominantly left ventricular cardiomyocytes differentiation of hiPSC-CMs in vitro. A human iPSC line was genome-edited to introduce a TBX5 - Cre /LoxP lineage tracing system. Genome-edited hiPSCs were differentiated and collected for scRNA-seq using the ICELL8 Smart-seq2 system. devCellPy models used for murine ventricular cardiomyocyte subtype predictions were applied for the prediction of the hiPSC-CMs. a Schematic of dual TBX5-Cre / MYL2-TdTomato lineage tracing system. b workflow for collection of day 15 ICELL8 data from genome-edited cell line. c Feature plots showing the expression of TurboGFP , LV marker HAND1 , ventricular markers (MYL3, MYL2, MYH7, MPPED2), and atrial markers ( NR2F1, KCNA5, VSNL1, STARD10) . d Left, Representative flow cytometry plot of cardiac troponin T (TNNT2) and TurboGFP expression at day 35 of cardiomyocytes containing TBX5 -lineage tracing system. Right, Quantification of GFP-positive percentage among TNNT2 + cardiomyocytes among 17 independent biological replicate differentiations. Error bars represent standard error around the mean. e Left, Representative flow cytometry plot of MYL2-TdTomato and TurboGFP expression at day 35 of cardiomyocytes. Right, Quantification of GFP-positive percentage among TdTomato+ cardiomyocytes among 17 independent biological replicate differentiations. Error bars represent standard error around the mean. f Predictions of cardiomyocyte subtypes for

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Experimental details: Mechanical stimulation partially improves tissue slice viability and structural integrity after 12 days in culture. a Bar graph shows quantification of the MTT viability of fresh heart slices (D0) or heart slices culture for 12 days either in static culture (D12 Ctrl) or in CTCM (D12 MC) ( n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) slices/group from different pigs, one way ANOVA test is performed; #### p < 0.0001 compared to D0 and ** p < 0.01 compared to D12 Ctrl). b Representative immunofluorescence images for troponin-T (green), connexin 43 (red), and DAPI (blue) for freshly isolated heart slices (D0) or heart slices cultured for 12 days under static conditions (Ctrl) or CTCM conditions (MC) (Scale bare = 100 um). Artificial intelligence quantification of the heart tissue structural integrity ( n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) slices/group each from different pig, one-way ANOVA test is performed; #### p < 0.0001 compared to D0 and **** p < 0.0001 compared to D12 Ctrl). c Representative images (left) and quantification (right) for heart slices stained with Masson's trichrome stain (Scale bare = 500 um) ( n = 10 slices/group each from different pig, one-way ANOVA test is performed; #### p < 0.0001 compared to D0 and *** p < 0.001 compared to D12 Ctrl). Error bars are representative of the Mean +- SD.

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Experimental details: Figure 2 Sodium Nitroprusside Enhances Generation of Cardiac Conduction System Cells (A) Expression of cardiac conduction system markers, HCN4 and Cntn2:EGFP, was examined by immunofluorescence staining using antibodies against either HCN4 or GFP. Scale bar, 200 mum. DMSO was used as a control. Arrows indicate cells that are double positive deriving from the SN-treated cultures. (B) Quantification of immunofluorescence staining of HCN4 + , Cntn2:EGFP + , and HCN4 + /Cntn2:EGFP + double-positive cells by MetaExpress image analysis. Results represent the combined data from three independent differentiation experiments. Error bars show SD. (C) Efficacy curve of SN in the Cntn2:egfp cell line. Results represent the combined data from three independent differentiation experiments. Error bars show SD. (D) Flow Cytometry analysis of Cntn2:EGFP expression. Following 5 days treatment of either SN or DMSO, cells were harvested at day 25 of differentiation. Quantification of Cntn2:EGFP expression is shown in the right panel. Results are from five independent experiments. Error bars show SD. (E) SN- or DMSO-treated cells co-express cardiomyocyte markers alphaMHC or TNNT2 with Cntn2:EGFP. Cntn2:EGFP + cells express relatively weak levels of CX43 (right panel). Scale bar, 200 mum. Asterisks indicate significant differences compared with DMSO. Statistical significance is indicated: * p < 0.05, ** p < 0.01, *** p < 0.001. See also Figure S2 .

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Experimental details: Figure 7 Inhibition of MEK Reverses the Hypertrophic Phenotype in BRAF-Mutant CMs (A) Dissociated EBs were treated with the MEK inhibitor U0126 and cellular area of cTNT + cells quantified. Exposure to U0126 (n = 130) significantly decreased the cellular area of BRAF-mutant CMs (n = 142) by 36% ( **** p < 0.0001), but had no significant effect (ns) on the cellular area of WT CMs (n = 57). Box-and-whisker plots show the median to the first and third quartiles and the minimum and maximum values. Scale bars, 200 mum. (B and C) Purified BRAF-mutant CMs exposed to U0126 displayed (B) decreased prevalence of irregular transients (n = 34) ( **** p < 0.0001) and (C) decreased SR Ca 2+ content (n = 12) ( *** p < 0.001). Data are presented as means +- SEM. n.s., not significant. Data represent two (WT2, 3) and three (BRAF1, 2, 3) biological and four technical replicates. See also Figure S7 .

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Experimental details: Figure 1 Structural characterization of H9-derived CMs. CMs were generated from H9 hESCs using the monolayer-based directed differentiation protocol. At day 20, CMs were immunostained for alpha -actinin (red) and cTnT (green). The cell nuclei were stained with DAPI (blue). Scale bar = 50 mu m.

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Experimental details: Figure 1. Generation of lamin A/C haploinsufficient hiPSC-CMs. (A) Predicted effect of the LMNA R225X mutation on the two splicing products lamin A and C. (B) Sanger sequencing of LMNA exon 4 in hiPSCs with heterozygous R225X mutation (top), or in hiPSCs obtained after CRISPR/Cas9-based scarless correction of the mutation (bottom). (C) Schematic of the protocol for step-wise directed differentiation of hiPSC-CMs. CHIR, CHIR-99021; AA, ascorbic acid. (D) Quantification of cardiac differentiation efficiency by flow cytometry for TNNT2 and NKX2-5 on hiPSC-CMs at day 14 of differentiation. (E) RT-qPCR analyses at the indicated stages of hiPSC-CM differentiation (see panel C). Differences versus mutant were calculated by two-way ANOVA with post hoc Holm-Sidak binary comparisons (*, P < 0.05; ***, P < 0.001; n = 3 differentiations; average +- SEM). (F) Representative Western blot for A- and B-type lamins and differentiation markers during iPSC-CM differentiation. (G) Quantification of lamin A/C expression in hiPSC-CMs from Western blot densitometries. Differences versus mutant were calculated by one-way ANOVA with post hoc Holm-Sidak binary comparisons (**, P < 0.01; ***, P < 0.001; n = 3 differentiations; average +- SEM). Throughout the figure (and in all other figures), Mut or Mutant indicates LMNA R225X hiPSCs, and Corr.1/2 or Corrected 1/2 indicates the two isogenic corrected control LMNA R225R hiPSC lines.

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Experimental details: Figure 1--figure supplement 1. Cardiomyocytes can be differentiated from iPSCs from both humans and chimpanzees. ( A ) Schematic representation of the protocol used to differentiate cardiomyocytes from human and chimpanzee iPSC lines (see Materials and methods for a detailed description). ( B ) Immunostaining of cardiomyocyte-specific markers in iPSC-CMs from a representative human and chimpanzee individual. The cytoplasmic markers TNNT2 (green) and ACTN1 (red) are shown in the left panel. The nuclear marker IRX4 (green), together with TNNT2 is shown in the right panel. Nuclei are stained with Hoechst. Staining of iPSC-CMs incubated with a fluorescently-tagged secondary antibody, without a prior incubation with primary antibody, is included as a negative control in the bottom panel.

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Experimental details: 1 Figure Serum progesterone concentration declines after birth and is associated with decrease in CM proliferation. A,B, Evaluation of CM proliferation by Ki67 and PH3 immunostaining at E16, P1, P7 and P14 hearts. Representative images with z-stacking are shown in A1 and B1, and quantification of percentages of Ki67 + and PH3 + CMs is shown in A2 and B2, respectively (n = 5). Scale bars are 20 um. cTnT indicates cardiac troponin T. C, Serum concentrations of progesterone in mice at E16, P1, P7 and P14 detected using ELISA kit (n = 5-8). *** P < .001 vs E16; ### P < .001 vs P1; &&& P < .001 vs P7. CM, cardiomyocyte

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Experimental details: 2 Figure Progesterone supplementation promotes postnatal CM proliferation in vivo. A, Overview of the experimental set-up of the daily intraperitoneal injection of progesterone (8 mg/kg) or control vehicle (corn oil) from P1 to P6. Hearts were harvested at P7. B-D, CM proliferation was evaluated by Ki67, PH3 and Aurora B immunostaining. Representative images with z-stacking are shown in B1, C1 and D1, and quantification of percentages of Ki67 + , PH3 + or Aurora B + CMs is shown in B2, C2 and D2, respectively (n = 6). Scale bars are 20 um. E, Representative images (E1) and quantification (E2) of the cell size of CMs (labelled by cTnT and pan-cadherin to show intact cardiomyocytes) isolated from P7 heart. Eighty to one hundred cells were randomly selected per heart (n = 6). Scale bars are 20 um. F, Heart weight and body weight (HW/BW) ratio (n = 10-11). G, The mRNA expression of cell cycle activators in mouse hearts was analysed by real-time PCR (n = 5). CM, cardiomyocyte

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Experimental details: 3 Figure Progesterone promotes CM proliferation in vitro in a progesterone receptor-dependent manner. Cultured P7 CMs were treated with control vehicle (DMSO) or progesterone (10 -7 M), alone or in combination with the progesterone receptor inhibitor RU486 (10 -6 M). CM proliferation was analysed 24 h after progesterone stimulation. A-C, CM proliferation was evaluated by Ki67, PH3 and Aurora B immunostaining. Representative images are shown in A1, B1 and C1, and quantification of percentages of Ki67 + , PH3 + or Aurora B + CMs is shown in A2, B2 and C2, respectively. (3000-5000 cells were randomly selected and analysed for each group, and four independent experiments were conducted). Scale bars are 40 um. D, The mRNA expression of cell cycle activators in P7 CMs was analysed by quantitative PCR (n = 5). ( ** P < .01, *** P < .001) vs Control; ( ## P < .01, ### P < .001) vs Progesterone. CM, cardiomyocyte

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Experimental details: 5 Figure Progesterone promotes CM proliferation through up-regulation of YAP expression. Cultured P7 CMs were treated with control (vehicle, DMSO) or progesterone (10 -7 M), alone or in combination with RU486 (A-C) or YAP siRNAs (D-F). A, Proliferation regulator mRNA expressions of CM were analysed by quantitative PCR (n = 4). B, Representative immunoblots (B1) and quantification (B2) of YAP protein expression were analysed by Western blotting (GAPDH was used as internal control) (n = 4). C, The mRNA expression of YAP target genes ANKRD1, CYR61 and CTGF was analysed by quantitative real-time PCR (n = 4). D-F, CM proliferation was evaluated by Ki67, PH3 and Aurora B immunostaining. Representative images are shown in D1, E1 and F1, and quantification of percentages of Ki67 + , PH3 + or Aurora B + CMs is shown in D2, E2 and F2, respectively (3000-5000 cells were randomly selected and analysed for each group) (n = 4). Scale bars are 40 um ( ** P < .01, *** P < .001) vs Control; ( ## P < .01, ### P < .001) vs Progesterone. CM, cardiomyocyte; YAP, yes-associated protein

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Experimental details: 7 Figure Progesterone promotes adult CM proliferation and improves cardiac function after MI. A, Adult mice were subjected to MI by ligation of the left anterior descending coronary artery and intraperitoneally injected daily with progesterone (8 mg/kg) or control vehicle (corn oil) from day 2 to day 35 post-MI. B-D, Hearts were harvested at day 7 post-MI; CM proliferation in the peri-infarcted area was evaluated by Ki67, PH3 and Aurora B staining in CMs. Representative images with z-stacking and quantification of percentages of those markers are shown (more than 5000 cells were randomly selected in each heart, and 6 mice were analysed in each group). Scale bars are 20 um. E,F, Echocardiographic (Echo) analyses of the effect of progesterone on cardiac function were performed 28 days after MI. Quantitative analysis of left ventricular ejection fraction (EF) and left ventricular fraction shortening (FS) is shown (n = 11-12). G, Hearts were harvested at day 35 post-MI. Representative images (G1) and quantification (G2) of fibrotic scar of heart sections were analysed using Masson's trichrome staining (n = 8). Scale bars are 1 mm. CM, cardiomyocyte; MI, myocardial infarction

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Experimental details: Fig. 2 Organization and microstructure of hECTs. Representative microscopy images of hECTs obtained along the horizontal axis of the hECT. a Single slice image obtained on a two-photon microscope, showing alpha-actinin (red) and collagen fibers (white), scale bar = 100 mum. b Confocal max projection showing Cardiac Troponin T (red), and nuclei stained with anti-histone H2B (green), scale bar = 100 mum. c Confocal max projection showing myofibril organization, with cardiac Troponin T (red), and nuclei stained with DAPI (blue,) scale bar = 5 mum

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Experimental details: Fig. 2 In vitro 2D and 3D cardiomyocyte cultures. (A) Timeline of the presented liquid marble procedure from start (0h) making the marble, until 96h after attachment of cardiosphere used for immunofluorescent staining. (B) 20-day-old cardiomyocytes grown in 2D were passaged to a glass coverslip and immunofluorescent staining after 96h of attachment shows expression of cardiac markers cTnT (green), NKX2.5 (red), a counterstain with Hoechst (blue) was performed. (C) Cardiospheres (indicated by the black arrow) were produced after 24h in a 50 µL Liquid Marble (LM) of different cardiac culture concentration (500, 1000 and 2000 cells/ ul). Phase-contrast pictures show attachment after cardiosphere harvesting after 24h and 96h. Maximal intensity projection images show cardiac marker expression for cTnT (green) and NKX2.5 (red) and nuclei (blue). Bar in phase-contrast images indicates 500 um, and 100 um in confocal images. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.) Fig 2

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Experimental details: Fig. 3 Purification of cardiac cultures using MACS. Phase contrast images show 2D cardiac cultures 48 h after passaging without purification and with MACS purification. Cardiac markers cTnT (green) and NKX2.5 (red) are showed in the immunofluorescent images, a counter stain for nuclei with Hoechst (blue) was performed. Bar indicates 500 um in phase-contrast images and 100 um in confocal images. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.) Fig 3