Figure 2. Genome-wide profiling of cardiac Charme^WT and Charme^KO transcriptomes.

(A) Heatmap visualization from RNA-seq analysis of Charme^WT and Charme^KO neonatal (PN2) hearts. Plot was produced by heatmap3 (Zhao, 2021). Expression values were calculated as FPKM, were log₂-transformed and mean-centered. FPKM, fragments per kilobase of transcript per million mapped reads. (B) Volcano plots showing differential gene expression from transcriptome analysis of Charme^WT vs. Charme^KO PN2 hearts. Differentially expressed genes (DEGs) validated through RT-qPCR (C) are in evidence. FC, fold change. (C) RT-qPCR quantification of upregulated (left panel) and downregulated (right panel) DEGs in Charme^WT vs. Charme^KO neonatal hearts. Data were normalized to Gapdh mRNA and represent means ± SEM of WT (n = 5) vs. KO (n = 4) independent biological pools (at least 3 littermates/pool). (D) Gene Ontology (GO) enrichment analysis performed by WebGestalt (http://www.webgestalt.org) on upregulated (left panel) and downregulated (right panel) DEGs in Charme^WT vs. Charme^KO pools of neonatal hearts. Bars indicate the top categories of biological processes in decreasing order of – log₁₀FDR. All the represented categories show a false discovery rate (FDR) value <0.05. (E) RT-qPCR quantification of pCharme targets in Charme^WT and Charme^KO extracts from E12.5, E15.5, and E18.5 hearts. DEGs belonging to the GO category ‘anatomical structure morphogenesis’ were considered for the analysis. Data were normalized to Gapdh mRNA and represent means ± SEM of WT and KO (n = 3) independent biological pools (at least 3 littermates/pool). (F) RT-qPCR quantification of the Myh6/Myh7 ratio in Charme^WT and Charme^KO extracts from E12.5, E15.5, and E18.5 and neonatal hearts. Data were normalized to Gapdh mRNA and represent means ± SEM of WT and KO (n = 3) independent biological pools (at least 3 littermates/pool). Schematic representation of the physiological Myh6/Myh7 expression trend is shown. (G) Upper panel: hematoxylin-eosin staining from Charme^WT and Charme^KO E15.5 cardiac transverse sections. Scale bars: 500 μm. Lower panel: quantification of the total area, the left and right ventricle cavities, and the thickness of the interventricular septum (IVS) in Charme^WT and Charme^KO E15.5 hearts. For each genotype, data represent the mean ± SEM of WT and KO (n = 3) biological replicates. (H) Representative images of Nppa (green) and TnnT2 (red) immunostaining in Charme^WT and Charme^KO (E15.5) cardiac sections. Regions of interest (ROI, orange squares) were digitally enlarged on the lower panels. Scale bar: 500 µm. Quantification of the area covered by the Nppa fluorescent signal is shown aside. Data represent the mean (%) ± SEM of WT (n = 4) and KO (n = 3) biological replicates. Data information: *p<0.05; **p<0.01, NS > 0.05, unpaired Student’s t-test.

Figure 2—figure supplement 1. Transcriptomic and phenotypic characterization of Charme^WT and Charme^KO hearts.

(A) Schematic overview of the workflow to identifying differentially expressed genes (DEGs) from Charme^WT and Charme^KO transcriptomes (left panel). Multidimensional scaling plot of leading fold change (FC) between each pair of Charme^WT and Charme^KO RNA-seq samples (right panel). Plot was obtained by using the plotMDS function from edgeR package (Chen et al., 2022). (B) Quantification by RNA-seq (FPKM) of upregulated (left panel) and downregulated (right panel) DEGs in Charme^KO vs. Charme^WT neonatal hearts. (C) Quantification by RNA-seq (FPKM) of pCharme and Charme-neighboring genes expression in Charme^WT vs. Charme^KO neonatal hearts. (D) Left panel: representative images for KI-67 (green) and DAPI (blue) stainings on Charme^WT and Charme^KO neonatal cardiac sections. Scale bars: 70 µm. Right panel: quantification of KI-67-positive nuclei/total nuclei on Charme^WT and Charme^KO cardiac sections from neonatal mice. Data are expressed as mean ± SEM of WT and KO (n = 4) biological replicates. (E) Left panel: RT-qPCR quantification of DEGs belonging to the ‘cell cycle’ GO class from Charme^WT and Charme^KO E15.5 hearts. Data were normalized to Gapdh mRNA and represent means ± SEM of WT and KO (n = 3) independent biological pools (at least 3 littermates/pool). Right panel: representative images for KI-67 (green) and DAPI (blue) stainings on Charme^WT and Charme^KO E15.5 cardiac sections are shown. Quantification of KI-67-positive nuclei/total nuclei on Charme^WT and Charme^KO cardiac sections from E15.5 mice. Data are mean ± SEM. of WT and KO (n = 3) biological replicates. Scale bars: 140 µm. (F) Western blot analysis for NPPA in Charme^WT and Charme^KO E15.5 cardiac extracts. β-ACTIN was used as a loading control. Quantification of NPPA signal intensity relative to β-ACTIN is shown below. Data are mean ± SEM of WT and KO (n=3) biological replicates. *p=0.03. A representative image is shown. (G) RT-qPCR quantification of trabeculae markers expression in Charme^WT vs. Charme^KO E15.5 cardiac extracts. Data were normalized to Gapdh mRNA and represent means ± SEM of WT and KO (n = 3) independent biological pools (at least 3 littermates/pool). (H) Representative image of Lectin (green) and TnnT2 (red) immunostainings in Charme^WT and Charme^KO E15.5 cardiac sections. Regions of interest (ROIs) (orange squares) were digitally enlarged on the lower panels. Scale bars: 500 µm. Quantification of the area covered by the Lectin fluorescence signal is shown aside. For each genotype, data represent the mean ± SEM. of WT and KO (n = 3) biological replicates. (I) Representative M-mode echocardiographic track of Charme^WT and Charme^KO 9–12 months aged mice. Quantification of heart morphology (EDD, end-diastolic diameter; ESD, end-systolic diameter) and function (FS: Fractional Shortening = (EDD-ESD)/EDD) were evaluated. Data represent the mean ± SEM of WT and KO (n = 10–13) biological replicates. Data information: *p<0.05, **p<0.01, ***p<0.001, unpaired Student’s t-test.