Extended Data Fig. 2: Dedifferentiated phenotypes are largely reversed in functionally redifferentiated hearts.
a,b, tOE/WT at all timepoints (a) and pOE/WT at Rediff (b) RNA expression fold change of ‘return to normal’ genes, involved in metabolism, proliferation and EMT-like features, determined by RT-qPCR. All values are normalised to their in-time point average WT value (black dashed line). n = 3 - 4 mice per group. c, Western blot quantification for data in Fig. 2b in order to validate the ‘return to normal’ behaviour of proteins involved in proliferation, EMT-like features and metabolism. n = 4 – 7 mice per group. d, Representative immunofluorescence images of isotype controls (WT) for the tOE hearts at Int. and Rediff timepoints shown in Fig. 2c, stained for Ki67, Nestin and Tomm20. Full quantification is provided in Fig. 2c. Scale bars = 50μm for Ki67 and NESTIN, 100μm for TOMM20. e’–f’’, Metabolic analysis of cultured P7 WT (n = 3) and OE (n = 5) CMs using an XFe96 Seahorse analyser. OCR (oxygen consumption rate) during the Cell Mito Stress Test (e’) and ECAR (extracellular acidification rate (glycolysis proxy)) during the Glycolysis Stress Test (f’). Maximal respiration/OCR (e’’) and Glycolysis (f’’). For (e’’) WT vs OE p = 0.0320. g, H&E-stained histological sections of WT Dediff and tOE Dediff, Int. and Rediff hearts. Images were acquired in the remote zones of MI injured hearts as a proxy for sham injury. Scale bars = 50μm. n = 3 for each group. h,i, Scatter plot of immune-related (h) and angiogenesis-related (i) GO term z-scores against enrichment significance (log10 p-value) for tOE/WT across all timepoints, based on Ingenuity Canonical Pathway Analysis of RNAseq data using a threshold fold change (FC) ≥ 1.5; adjusted p ≤ 0.05. Arrows on each line indicate the direction of the GO term from Dediff to Int. to Rediff. Z-scores below −2 are predictive of pathway inactivation and above +2 are predictive of pathway activation. Values above the horizontal dashed black line represent statistically significant enrichment. j,k, Heatmaps of differentially expressed genes corresponding to the IPA analysis in (i), (j) and an independently curated list of angiogenesis genes (k), compiled as described in Fig. 1g. l, Western blot quantification for data in Fig. 2g in order to validate the proteins involved in metabolism, cytoskeletal signalling and heart function that remain differentially expressed at Rediff. In all panels numerical data are presented as mean ± SEM; statistical significance was calculated using two-tailed unpaired Student’s t-test in (a–c,e’’,f’’,l) between the in-time point WT and tOE values. *p ≤ 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
