Dbh+ catecholaminergic cardiomyocytes contribute to the structure and function of the cardiac conduction system in murine heart

Figures

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  Fig. 1. Single-cell RNA sequencing identified a Dbh+ subpopulation within the cellular landscape of the murine cardiomyocyte lineage. a Summary of workflow: whole embryos (E8.5, E10.5) or whole hearts (E12.5, E14.5, E16.5, P3) were sampled for single cell RNA sequencing. Slices of whole heart from E12.5, E14.5, and P3 were sampled for Stereo-seq spatial transcriptomics. We used 10X Genomics and Stereo-seq for sequencing. We performed quality control before we clustered and labelled cells based on their gene expression. We performed a range of downstream analyses with both single-cell RNA sequencing and Stereo-seq data. b PHATE plot of the cellular landscape of the cardiomyocyte lineage. Numbered labels indicate the cell type with the corresponding colour on the PHATE plot, with full names below. (See Supplementary Data 2 for interactive version). c PHATE plot of the Dbh+ cardiomyocyte subpopulation in the developing murine heart. Numbered labels indicate the cell type with the corresponding colour on the PHATE plot, with full names below and are common with panel b. (See Supplementary Data 3 for interactive version). d Heatmap demonstrating selected marker genes for each population…

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  Fig. 2. Dbh is expressed dynamically across the developing heart, as resolved with nanoscale-resolution Stereo-seq of a DbhCre/Rosa26-tdTomato reporter line. a Schematic of DbhCre/Rosa26-tdTomato reporter line creation and analysis for spatially-resolved transcriptomics with Stereo-seq. b Plot of spatially-resolved transcriptomic pixels (each pixel consists of 20 × 20 from spots of individual diameters between 500 or 715 nm) identified across hearts from E12.5, E14.5, and P3 mice, coloured by cell type identified through unsupervised clustering with corresponding cell types coloured below. c (i) Dot plot describing Dbh and Wpre (a tdTomato construct-derived transcript) expression, with other key marker genes, across major cardiomyocyte populations identified through unsupervised clustering of E12.5, E14.5, and P3 Stereo-seq data. Dots are sized by percentage of cells expressing each respective gene in each respective cell type. Dots are coloured by average expression of the respective gene in the respective cell type, with yellow indicating higher expression, and purple lower expression. (ii) Spatially-resolved plots describing Dbh and Wpre (a tdTomato-derived transcript) expression,…

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  Fig. 3. Prospective fate mapping analysis of DbhCre/Rosa26-tdTomato heart throughout the developmental stages. a Representative gross images of tdTomato-expressing signals from E10.5 to E12.5 mouse embryos (left 3 panels) and an image of tdTomato-expressing signals of a sagittal section of a E12.5 embryo (far right panel). b Representative images showing tdTomato signals in RA and SV region of two sagittal section of E12.5 embryos (i) and magnified RA area (ii). c Representative RNAscope images showing the distribution of Bmp10 (green), CCS markers Cacna2d2 (magenta) in a whole field of E12.5 heart, indicating the anatomy of right atria, trabecular region and main CCS regions. d Representative RNAscope images showing the distribution of Dbh (green), CCS markers Id2(magenta) and Cacna2d2 (magenta) in whole field images of E12.5 heart respectively, unveiling the overlap expression in AVN, SV HIS and PKJ. Scale bar: 200 μm. Red Arrowed: tdTomato-expressing cells SG Sympathetic Ganglia, SV Sinus Venosus, RA Right Atrium, V Ventricle, AVN Atrioventricular nodal, HIS His-bundle branch, PKJ Purkinje fibre network, n > =3 hearts/slices for each stage.

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  Fig. 4. Fate mapping of Dbh+-derived cardiomyocytes in the developing and postnatal hearts by using DbhCre/Rosa26-tdTomato reporter. a Representative images of immunostaining showing the co-expression of tdtomato (red) and α-actinin (green) illustrating the distribution of Dbh+-derived CMs in HIS and trabecular regions at E14.5. Scale bar: 100 μm. b Representative image of tdTomato expressing cells in a four-chamber view section of P3 heart. Scale bar: 100 μm. c Representative images of immunostaining showing the co-expression of tdTomato (red) and α-actinin (green), illustrating the distribution of Dbh+-derived cardiomyocytes in RA, HIS, LPF and RPF regions in a heart at P3. Scale bar: 50 μm. d Representative images of immunostaining showing the co-expression of tdTomato (red) and α-actinin (green) illustrating the distribution of Dbh+-derived cardiomyocytes in AVN, HIS, PKJ regions in an adult heart. Scale bar: 100 μm, 100 μm, 10 μm, 10 μm (From left to right). e The distribution profile of Dbh+-derived CMs and multi-angle hyperspectral three-dimensional reconstruction results for Dbh+-derived CMs in a P3 heart sections. All results were written into VTK file format. f The distribution…

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  Fig. 5. Validation of spatial distribution of Dbh+-CMs and their close association with CCS. a Representative images of immunostaining with anti-Flag (red) and anti-CFP (red), plus α-actinin (green) antibodies showing the expression of Dbh+-CMs in HIS region at E14.5 and adult (P56) by using DbhCFP reporter. b Representative images in AVN and HIS with α-actinin (green) immunostaining showing the expression pattern of Dbh+-CMs by using DbhCreERT/Rosa26-tdTomato inducible reporter at P56. c Representative images showing co-localization of Dbh+-derived CMs and Cx40+ cells in PKJ network in both left and right ventricle in DbhCre/ChR2-tdTomato/Cx40-eGFP neonatal mouse hearts. Scale bar: 50 μm n = 3 hearts/slices for each stage.

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  Fig. 6. Direct optogenetic assessment of Purkinje fibre function implicates Dbh+-derived CMs in the ventricular CCS. a Representative ECG traces from Langendorff-perfused hearts subjected to localized light pacing in four different chambers and 6 different subregions of hearts (SR: sinus rhythm, LA: left atrium, RA: right atrium, RVbase: right ventricle base, LVbase: left ventricle base, RVapex: right ventricle apex, LVapex: left ventricle apex) with blue light isolated from optogenetic transgenic mouse lines expressing the fused protein tdTomato-ChR2 under the control of the promoters for MHC (MHC-ChR2), Dbh (Dbh-ChR2) and Cx40 (Cx40-ChR2). Blue triangles indicate when blue light pacing was applied. b A diagram displaying the electrical responsiveness of the different heart chambers to similar regional optical pacing across the different mouse genetic lines. c Comparison of effective refractory periods (ERP) determined by RV epicardial optical programmed stimulation in hearts from three optogenetic transgenic mouse lines. (n = 5 for MHC-ChR2 model; n = 7 for Dbh-ChR2 model; n = 5 for Cx40-ChR2 model). d Representative ECG traces of ectopic beats originated by epicardial light…

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  Fig. 7. Electrophysiological characterization of Dbhcko and Dbhf/f hearts reveals the role of cardiomyocyte Dbh expression in intracardiac conduction. a Typical ECG traces of isolated hearts at baseline from Dbhcko and Dbhf/f mice. b Representative ECG traces for evaluation of AVNERP by programmed electrical stimulus (PES), the drop of S2 induced ventricular QRS complex is highlighted in the grey box indicating the AVNERP. c Consecutive S1S1 PES pacing protocol was conducted to measure the Wenckebach point at 2:1 conduction, the highlight part indicates the Wenckebach point—that only one QRS complex was generated by two S1 stimulations. d The statistical comparison of P-R, RR intervals, AVNERP and Wenckebach point between Dbhcko and Dbhf/f hearts. n = 6 per group. Both genders of mice aged 8–12 weeks are utilized in ex vivo ECG studies. Statistical results analysis was performed using GraphPad Prism 8, and statistical significance was calculated by a Student t test.

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  Fig. 8. Transmission electron microscopy identifies catecholaminergic vesicles within CMs. a, b Transmission electron microscopy (TEM) imaging: high electron density vesicles (arrowed) detected in cardiac myocytes. c Transmission electron microscopy (TEM) imaging: high electron density vesicles (arrowed) detected in adrenal medullary chromaffin cells. d The selected region for EDS analysis of chromium. Weak chromium signals measured as CrKa and CrKb peaks in EDS spectrum were detected in cardiomyocyte in contrast to the strong chromium signals in adrenal medullary chromaffin cells. Nu, cell nuclei. Scales bar: 8b: left: 2 µm, right: 500 nm; 8c: left: 2 µm, right: 500 nm; 8d: left: 1 µm, right: 500 nm; n = 3 hearts.

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  Fig. 9. Proposed relationships of SN innervation and Dbh+-CCS formation. a Dbh+-CM-associated CCS formation occurred concurrently with sympathetic innervation from E12.5 after neural crest cells migration occurred in SN ganglia and heart region from E10.5. Such process is in parallel to the processes of ventricular trabeculation and chamber formation. b From E14.5 onwards, the Th positive nerve fibres were clearly identified and they extended to the AV junction, His-Bundle and part of the ventricular myocardium. Such a process occurred concurrently with Dbh+-CM-associated CCS formation. c E16.5 onwards to postnatal, SN innervation continues and CCS is maturing. d From postnatal to adulthood, SN innervation and maturation of CCS are completed. e Illustrating the relationship between Dbh+ cells, Dbh-derived cardiomyocytes, and Dbh+-CMs: Dbh expressing cardiomyocytes.