The G4 resolvase Dhx36 modulates cardiomyocyte differentiation and ventricular conduction system development

Figures

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  Fig. 1. Dhx36 protein expression in embryonic and adult hearts. a Immunohistochemical (IHC) analysis of ED10.5, ED11.5, and ED12.5 embryonic hearts stained with IgG (upper panels) or α-Dhx36 (lower panels). Panel i presents a magnified view of a specific region (boxed area) within the left ventricle of an ED12.5 heart. b IHC staining of adult hearts with IgG (left upper panel) or α-Dhx36 (left lower panel). Panels i and ii show magnified views of the corresponding boxed areas. c Western blotting showing Dhx36 expression in cytoplasmic (Cyt.) and nuclear (Nuc.) subcellular compartments of total adult hearts. Specific expression was validated by analyzing total extracts of C2C12 myocytes and mouse brain. Anti-tubulin (Tub.) was blotted in the same membrane after α-Dhx36 and served as a cytoplasmic control. Samples derived from the same experiment. d IHC of Dhx36 expression in WT and Dhx36 Nkx2-5 ED12.5 embryonic hearts (top left panels). The right panels depict magnifications of regions of the right ventricles (RV, top) or left ventricles (LV, bottom). As a control for Dhx36 staining, the bottom left panels show IHC in the neural tube of the same WT and Dhx36 Nkx2-5 embryos. All…

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  Fig. 2. Dhx36 deletion induces dilated cardiomyopathy, reduced ejection fraction, and surface ECG alterations. a Kaplan-Meier survival curve comparing WT mice (Dhx36 f/f) with Dhx36Tnnt2 mutant mice. b Gross morphology of hearts from 21-day-old WT (top left) and Dhx36Tnnt2 conditional KO (cKO.1) mice (bottom left). Right panels show hematoxylin & eosin (H&E)-stained sections of the corresponding hearts. c Left panels: a 90-day old Dhx36Tnnt2 mouse (cKO.2) exhibiting overt signs of dilated cardiomyopathy and non-compaction of the left ventricular (LV) myocardium. The left atrium displays a prominent thrombus in the H&E-stained section. Right panels: picrosirius red stained sections highlight larger LV fibrotic areas (lower) compared to representative WT (upper) sections. The pictures in b and c are representative of 5 and 3 independent experiments, respectively, analyzing WT and mutant hearts of around similar ages, all showing similar phenotypes. d Box plots representing the distribution of IQR values for LV ejection fractions (LVEF; left) and the LV end-diastolic (LVd) volumes (right) of Dhx36Tnnt2 mutant mice (n = 10) and WT (n = 10). Lower and upper hinges of the boxes correspond to…

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  Fig. 3. Dhx36 deletion increases abnormal paroxysmal arrhythmic events. a, b Comparative analysis of RR intervals (heart rate calculated as [1000/RR interval in ms] x 60) between Dhx36Tnnt2 mutant mice and WT (a), and Dhx36Myh6 mutants and WT (b). c Incidence of relevant atrioventricular (AV) block events during ECG recordings of WT mice and Dhx36Tnnt2 and Dhx36Myh6 mutant mice. d Representative ECG tracings depicting a WT mouse and a Dhx36Tnnt2 mutant mouse with a paroxysmal AV block event. e Incidence of sinus node dysfunction during ECG recordings in WT mice and Dhx36Tnnt2 and Dhx36Myh6 mutant mice. f RR variability representation of Dhx36Tnnt2 mice (in light red) as compared to WT mice (in grey). Each dot represents a RR interval. g Sample tracing with overt signs of sinus node dysfunction (arrowhead) during the ECG recording of a Dhx36Tnnt2 mutant mouse. h Incidence of premature ventricular complexes (PVC) during ECG recordings in WT mice and Dhx36Tnnt2 and Dhx36Myh6 mutant mice. i Sample tracing of a potential nonsustained bidirectional ventricular tachycardia episode in an ECG recording of a Dhx36Tnnt2 mutant mouse. Green arrowheads show one PVC morphology, probably from Purkinje…

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  Fig. 4. Impact of perinatal and embryonic Dhx36 deletion on CCS morphogenesis. a Whole-mount (WM) confocal immunofluorescence (IF) of hearts of 41-day-old (PD41) WT and Dhx36Myh6 littermates, revealing an underdeveloped ventricular conduction system/Purkinje fiber (VCS/PF) network in the mutant. The pictures are representative of 3 independent experiments, analyzing WT and mutant hearts of similar ages, all rendering similar results. b Left: gross morphology of hearts from PD28 WT and Dhx36Tnnt2 littermates. Right: WM confocal IF of the same hearts, indicating an undetectable VCS/PF network in the mutant. Representative images are presented from a total of three Dhx36Myh6 and five Dhx36TnnT2 analyzed mutants. The pictures are representative of 5 independent experiments, analyzing WT and mutant hearts of similar ages, all rendering similar results. c–g ISH analysis of PD7 (P7) wild type and Dhx36Tnnt2 hearts hybridized with various CCS markers. Representative images are shown, three wild type and mutant embryos were examined; rv, right ventricle; lv, left ventricle. Scale bars, 100 µm in a, b (confocal images), 1 mm in b (whole hearts images). 200 µm in (c–g, general heart views), 100 µm…

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  Fig. 5. Single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq multiome of neonatal WT and Dhx36Tnnt2 mutant hearts. a Multimodal Uniform Manifold Approximation Projection (UMAP) visualization of cell clusters in WT hearts, randomly colored by identity. b Heatmap illustrating expression of Dhx36 in each cell cluster, projected on the UMAP graph of WT heart. c Violin plots presenting expression of Dhx36 in individual cell clusters. d Multimodal UMAPs displaying all WT cells in blue (n = 6499) and cKO cells (n = 5426) in red (upper panel). Below, WT and KO cells are separated in their multimodal UMAPs, randomly colored by identity as in (a). e, Fraction (%) of cell population clusters in each sample. Refer to Supplementary Fig. 6 for additional details. EC coronary = Coronary endothelial cells; EC Endoc.= Endocardial endothelial cells; SMCs= Smooth muscle cells.

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  Fig. 6. Single-nucleus RNA sequencing (snRNA-seq) and snATAC-seq multiome identify differences between WT and mutant hearts in cardiomyocyte populations. a Zoomed UMAP visualization of cardiomyocyte clusters colored by identity in the WT (left) and the mutant heart (right). b Violin plots depicting the expression of specific representative genes for each cardiomyocyte subcluster. Refer to Results for definition of CM clusters.

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  Fig. 7. Widespread transcriptomic alterations of mutant versus WT cardiomyocytes. a Quantitative PCR of selected cardiac conduction system genes and genes deregulated in the snRNA-seq between WT (n = 3) and mutant neonatal PD7 (KO; n = 3) hearts. b CTCF motif enriched at mutant cardiomyocyte-active open chromatin regions compared to WT (left). UMAPs of a zoom of the KO and WT cardiomyocytes, depicting their level of open chromatin at CTCF motifs (right). Violin plots of Z scores of CTCF open chromatin in specific CM1/mCM0, CM2/mCM2 and auricular CM7/mCM7 CM-specific clusters are shown below. Inset boxplots show the median, lower and upper hinges as well as whiskers as in Fig. 2d and i. Significance was determined in a using unpaired two-sided t-test; the data are presented as mean ± SEM with the p-values inserted. Source data for a and b are provided in the source data file.

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  Fig. 8. G4 structures and transcriptional alterations of Dhx36 gene targets. a Examples (Ntn1 and Nr4a1) of genes downregulated in KO CMs (Supplementary Data 10) with G4s overlapping open chromatin in their promoters (TRUE). b Example (Esrrb) of a gene downregulated in KO CMs with G4 not overlapping open chromatin in its promoter (FALSE). c Example (Rai2) of a gene upregulated in KO CMs with G4s overlapping open chromatin in their promoters (TRUE). On the left, the plots show the ATAC-seq data with the peaks of open chromatin in the promoter regions of the genes in all ventricular WT CMs (CM1) or mCM0, in WT + KO atrial CMs (CM7), and in VCS/PF (CM6) cardiomyocytes. The corresponding gene (with accessible peak represented as a grey box), the TRUE G4 (blue colored box) or FALSE G4 (salmon colored box) and the links between the regulatory regions (lower) are also shown. On the right, the violin plots of RNA-seq expression of the genes are shown in each cluster.

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  Fig. 9. G4-resolvase-dependent transcriptional activation of Dhx36 CM target genes. a Luciferase transcriptional activation assays in HeLa cells of promoter regions of Dhx36, Kcne1, Bcl2l11, Ntn1, Ppargc1a (Pgc1a), Fhl2, and Nr4a1 cloned in pGL4.25 luciferase expressing vector. The luciferase activity, measured in Relative light units (R.L.U.) of the different constructs, are shown in the presence or absence of 20 μM of the G4 stabilization drug TMPyP4. b same as in (a) with Dhx36, Drd2, Hcn4 and Cntn2 promoters. The pGL4.25 vector, presenting a minimal promoter, is shown as a negative control. Significance was determined in a and b using unpaired two-sided t-test; the data with the p-values included are presented as mean ± SEM (n = 3). The experiments shown in (a) and (b) are representative of 3 independent experiments (total n = 9), with similar results. Source data are provided in the source data file.

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  Fig. 10. Model of the impact of Dhx36 deletion in the heart. Cardiac phenotypes observed upon Dhx36 deletion with the indicated Cre-driver lines described in this study and in Nie et al8 and Huang et al9. For each mutant line, the dotted red line indicates the time window of Cre-driver allele action, and red shading indicates the expected anatomical recombination domain and deletion strength within the heart. Timelines depict the approximate extent of survival, the onset of lethality, and the phenotype penetrance for each driver. The panel to the right indicates the range and intensity of the cardiac phenotypes reported here and in previous studies. AVB, atrioventricular block; CCS, cardiac conduction system; LA, left atrium; LVNC, left ventricular non-compaction; RV, right ventricle; RVNC, right ventricular non-compaction; VCS, ventricular conduction system.