Extended Data Fig. 3: In vitro and in vivo analysis of cardiomyocyte-specific GR Knock Out (GR-cKO) mouse model.
(a) Immunofluorescence analysis of GR in cardiomyocyte cell cultures isolated from postnatal day 1 (P1) GR-cKO and control mice (403 CMs analyzed from n = 6 littermate mice sacrificed in a single session), showing the absence of GR expression in cardiomyocytes isolated from GR-cKO mice; arrows point at GR-positive and GR-negative nuclei in controls and GR-cKO cardiomyocytes, respectively; scale bars, 45 μm; (b) RT-PCR analysis of markers of cardiac pathological hypertrophy (Nppa, Nppb and Acta1) in postnatal day 7 (P7) GR-cKO and control heart lysates (n = 8 mice); (c) In vivo cardiomyocyte sarcomere status evaluation by immunofluorescence analysis of cardiac troponin T (cTnT) in P7 control (ctrl) and GR-cKO heart sections; images were obtained using a confocal microscope (n = 6 mice); scale bars, 15 μm for the main picture and 5 μm for the magnification; (d-g) Ultrastructure analysis by transmission electron microscopy (TEM) of P1 control (d,e) and GR-cKO (f,g) heart sections (n = 6 murine hearts isolated in two independent experiments), showing a slight more immature myofibrillar architecture in GR-cKO cardiomyocytes, characterized by less numerous myofibrils, sometimes randomly organized with cellular substructures (dotted lines in f compared to d), and isolated mitochondria (yellow arrows in f compared to d and magnification in g compared to e). Scale bars, 2 μm in (d, f) and 1 μm in (e, g). Legend for electron microscopy images: N = nucleus. (h) Heatmap of the top 5 statistically significant differentially expressed genes by RNA-Seq transcriptome analysis of P7 GR-cKO versus control hearts (n = 6 mice); the most statistically significant downregulated gene is Nr3c1 (GR); (i) Expression of de-differentiation markers (Runx1, Dab2 and Kit) in GR-cKO vs control P7 hearts from RNAseq analysis (n = 6 mice); (j) Cardiomyocyte dedifferentiation analysis by immunofluorescence staining for RUNX1/cTnT of P7 control (ctrl) and GR-cKO heart sections (n = 6 mice with a total of 17746 CMs analyzed); representative images are provided; scale bar, 20 μm; arrows point at dedifferentiated cardiomyocyte. In panel b and j, numerical data are presented as mean (error bars show s.e.m.); statistical significance was determined using two-sided Student’s t-test.
