Fig 2: Yap influences a transcriptional programme that impacts skeletal muscle metabolic substrate utilisation.A Schematic demonstrating integrated transcriptomics and proteomics experimental design including PCM1 cell sorting strategy to isolate adult myonuclei from 8-week-old C57BL/6 male mouse limb muscles at 28 days following treatment with AAV6:lacZ-shRNA or AAV6:Yap-shRNA, (B) MA plot of ATAC-sequencing showing more and less accessible regions of chromatin following Yap knockdown. Differential peaks are shown in red (n = 3 biologically independent animals; adjusted p value < 0.01, multiple comparisons were corrected using Benjamini–Hochberg procedure), (C) MA plot of RNA-sequencing showing up and downregulated transcripts following Yap knockdown. Differential transcripts are shown in red (n = 3 biologically independent animals; adjusted p value < 0.01, multiple comparisons were corrected using Benjamini–Hochberg procedure), (D) top five upregulated and (E) top five downregulated GSEA terms from RNA-seq data generated in (C), (F) volcano plot of proteins detected using label-free proteomics in muscles treated as in (A). Grey dots show proteins that were not significantly different, blue dots show proteins that were significantly downregulated and red dots proteins that were significantly upregulated (n = 5 biologically independent animals; adjusted p value < 0.01, t test with correction for multiple comparisons using Benjamini–Hochberg procedure), (G) GSEA analysis of proteomics datasets. Top five upregulated terms are shown in red, top five downregulated terms in blue.

Fig 3: Tregs in the maternal serum promote CM proliferation. a Rat neonatal CMs were exposed to the serum collected from neonatal (NS), adult (AS), pregnant (PS) and Treg-depleted pregnant (TDPS) animals and proliferation measured by EdU incorporation. Nuclei are stained blue with 4’-6-diamidino-2-phenylindole (DAPI) and CMs green by anti-a-actinin antibodies. Red nuclei indicate EdU incorporation. Scale bar, 100 µm. Insets shows a higher magnification of the area defined by the white dotted line. b Quantification of EdU+ CM nuclei (% of total CMs) after exposure to the different sera. c Outline of the experimental procedure followed for Treg depletion in vivo in pregnant animal. The Treg-depleting agent, either PC61 antibodies in CD1 mice or Dyphteria Toxin (DT) in DEREG mice, was administered i.p. to the pregnant mother at embryonic days 10 (E10) and 15 (E15). EdU was administered i.p. every day from E10 to E18, when embryos were removed for histological analysis. d Quantification of EdU+/ a-actinin CM nuclei (% of total CMs) in the heart of embryos harvested from either control (white bars) or Treg-depleted (gray bars) mothers, in the two models of Treg depletion described in panel (c). e Longitudinal section of a whole E18 heart, in which nuclei are stained blue with DAPI, CMs green by anti-a-actinin antibodies and EdU incorporation is shown in red. Scale bar, 1 mm. f High magnification images of the indicated regions of embryonic hearts from control (C) and Treg-depleted (TD) mothers, in which nuclei are stained blue with DAPI, CMs green by either anti-a-actinin or PCM1 antibodies and EdU incorporation is shown in red. RV: right ventricle, LV: left ventricle. White numbers indicate the percentage of EdU+ CMs in each cardiac region. Scale bar, 20 µm. Values in (b) and (d) are mean ± s.e.m., n = 3 biological replicates. One-way analysis of variance and Bonferroni/Dunn’s post hoc tests were used to compare multiple groups (b). Pairwise comparison was performed with the Student’s t-test (d). **P < 0.01, ***P < 0.001 relative to control

Fig 4: Gating strategy for Isolation of Diploid and Triploid Cardiomyocyte NucleiRepresentative flow cytometry plots for immunolabeling of pooled heart samples collected at P4 with antibody against PCM1 to label cardiomyocyte nuclei and Hoechst for DNA staining.(A–D) Unstained sample (A) and samples only stained for PCM1 (B) or Hoechst (C) were used as negative controls for gating. 2n cardiomyocytes and 4n cardiomyocytes are identified in samples stained with both Hoechst and PCM1 antibody (D). The different fluorescent intensities (y-axis) of Hoechst staining indicate the difference in DNA ploidy (2n vs 4n).

Fig 5: Altered cellular tissue composition in the LV myocardium of adult mice on LPD. (a) The number of cardiomyocyte and non-myocyte nuclei per tissue area was determined in LV myocardium of adult SPD and LPD mice based on immunofluorescence staining for PCM1 (red). WGA and DAPI staining was used to label cell membranes (green) and nuclei (blue), respectively. The total number of nuclei as well as the number of PCM1 positive cardiomyocyte nuclei per area was increased in LPD compared to SPD hearts. The number of non-myocyte nuclei per area as well as the ratio of non-myocyte/cardiomyocyte nuclei were not different between groups (SPD n = 3, LPD n = 5 litters, *P < 0.05, **P < 0.01). (b) Fibroblasts and myofibroblasts were quantified in the LV myocardium of adult LPD and SPD hearts based on immunofluorescence staining for vimentin and smooth muscle actin (SMA), respectively. The number of vimentin positive cells normalized to the total number of nuclei was reduced in LPD compared to SPD hearts, whereas SMA positive cell numbers were unchanged (n = 4 mice per group, *P < 0.05).