Development of autonomic innervation at the venous pole of the heart: bridging the gap from mice to human

Figures

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  Fig. 1. Embryology of cardiac autonomic innervation. A Cardiac autonomic nerves at the arterial and venous poles are primarily derived from neural crest cells (NCCs; dark blue dots), multipotent cells migrating from the neural tube region. Parasympathetic nerves emanate from the cranially situated cardiac (vagal) NCCs while sympathetic nerves originate from trunk NCCs. B Cardiac (vagal) NCCs, precursors of parasympathetic nerve cells, migrate along the developing vagal nerve pathway directly towards the heart to form the cardiac parasympathetic ganglia. From there, parasympathetic nerve projections extend to the venous pole. Trunk NCCs, precursors of sympathetic nerve cells, migrate ventrally towards the dorsal aortae to form the paravertebral sympathetic trunks. From there, sympathetic nerves extend into the heart through the cardiac plexus alongside the previously developed vagal nerves. B is modified after Vegh et al. [1], published open access under Creative Commons Attribution License (CC BY)

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  Fig. 2. Examples of normal heart rhythm and ectopic heartbeats in wildtype mouse embryos. A Pulsed-wave Doppler tracing across the mitral and aortic valves showing normal heart rhythm at E18.5. During each cardiac cycle, there is a biphasic ventricular inflow pattern (E’ and A’) followed by a monophasic ventricular outflow pattern. B Pulsed-wave Doppler tracing at E13.5 showing multiple premature atrial contractions (PAC, yellow arrows), i.e. extrasystoles, all followed by compensatory pause [illustrated by the elongated passive filling phase (red arrows)]. C Pulsed-wave Doppler tracing at E18.5 showing a PAC followed by a tachycardia with a monophasic ventricular inflow pattern (only A’). E’: passive ventricular inflow due to ventricular relaxation; A’: active ventricular inflow due to atrial contraction; PAC: premature atrial contraction; S’: ventricular outflow due to ventricular contraction

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  Fig. 3. Prenatal time trends of HRV parameters in murine and human embryos/fetuses. Time trends using linear mixed models corrected for mean embryonic/fetal heart rate and sex are provided separately for all HRV parameters. The dotted lines indicate 95% confidence intervals (CI) and the black dots indicate the raw data points. A–E HRV parameters in mouse embryos. A In mouse embryos, mean heart rate increased throughout gestation. B, C SDNN consistently low, whereas RMSSD demonstrated a slight decreasing trend. This suggests that, although a trend towards a decrease in parasympathetic activity during development was observed, definitive establishment of cardiac autonomic function is not yet observed. D The SDNN/RMSSD ratio remained predominant to RMSSD (i.e. ratio < 1), but increased in favor of SDNN throughout gestation. E pNN10 showed no specific trend. F–J HRV parameters in human fetuses. F In human fetuses, mean heart rate decreased throughout gestation. G–H SDNN and RMSSD increased from 20 to 30 WGA, suggesting that cardiac autonomic activity (mainly parasympathetic activity) establishes at approximately 20 WGA and matures until 30 WGA. I The SDNN/RMSSD ratio remained predominant to…

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  Fig. 4. Neural crest cell distribution at early cardiogenesis (i.e. cardiac looping phase) in mice and humans. A–D Mouse sections at 9.5 depicting Wnt1Cre-GFP+ neural crest lineage (green) cell distribution at the venous pole. The approximate location of B–D is indicated by the white dotted lines in A. Note C and D are from different sections. A’–D’ Human sections at 6 + 4 WGA showing the distribution of HNK-1+ (neural crest) cells (green) at the venous pole. The approximate location of B’–D’ is indicated by the white dotted lines in A’. Note D’ is from a different section. A, A’ Overview sections in mice (A) and humans (A’). Note that in both species neural crest (-derived) cells are prominently present in the regions of the primary sympathetic chains adjacent to the dorsal aortae (red arrows) and in the mesenchyme surrounding the foregut where the parasympathetic cells and vagal nerves develop (blue arrows). Nota bene: endothelial cells (PECAM-1) are also stained in A (white). B-C, B’-C’ At this early stage, no expression of neural crest cells (NCCs) is discerned in the putative right-sided sinoatrial node (SAN; yellow dotted line in B, B’) and left-sided SAN (C, C’, yellow dotted…

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  Fig. 5. First parasympathetic nerves at the venous pole in proximity to the developing pulmonary vein in mice and humans. A, A’ At E11.5 in mice (A) and 6 + 4 WGA in human (A’), a small number of neural crest(-derived) cells (Wnt1Cre-GFP+ in mice and HNK-1+ in humans) are encountered in the dorsal mesocardium and sinus venosus myocardium nearby the developing pulmonary vein (blue arrowheads). Also note the high amount of NCCs at the sympathetic chains (red arrows) and area of the vagal nerve (red asterisks). B–D, B’–D’. In both species, the sympathetic chains (red arrows) stain positive for TUBB3 (B, B’; yellow), ChAT (C, C’; magenta) and TH (D, D’; cyan). Also the vagal nerves have developed (red asterisks), staining positive for TUBB3 (B, B’) and ChAT (C, C’) with minimal co-expression of TH (D, D’). The first parasympathetic nerves are identified in the dorsal mesocardium nearby the developing pulmonary vein (red arrowheads), which stain positive for TUBB3 (B, B’) and ChAT (C, C’) but are negative for TH (D, D’). Scalebars: 100 μm. Br: pulmonary bronchus; dAo: dorsal aorta; MPES: midpharyngeal endothelial strand; Oe: oesophagus; LCV/RCV: left/right cardinal vein; LV/RV: left/right…

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  Fig. 6. Anatomical differences at the venous pole at mid-cardiogenesis between mouse and human. A, B Mouse sections at E12.5 showing that the sinus venosus is connected laterally to the right- and left cardinal veins. Note that the transient left-sided SAN is still present at this stage (red arrow) and the myocardialization of the right cardinal vein is initiated. A’, B’ Human sections at 9 WGA depicting that the left superior cardinal vein has regressed. Its remnant, the ligament of Marshall (black arrow), is well discernable at this stage and shows high expression of HNK-1. Scalebars: 100 μm. AoV: aortic valve; Br: pulmonary bronchus; dAo: dorsal aorta; LA/RA: left/right atrium; LCV/RCV: left/right cardinal veins; LM: Ligament of Marshall; LV/RV: left/right ventricle; Oe: oesophagus; SAN: sinoatrial node

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  Fig. 7. Neural crest and autonomic nerve markers surrounding the pulmonary vein and dorsal mesocardial protrusion in mouse and human at mid-cardiogenesis. A–D Mouse sections at E12.5. A’–D’ Human sections at 8 WGA. A, A’ Clusters of neural crest (-derived) cells (Wnt1Cre-GFP+ in mice and HNK-1+ in humans) surround the orifice of the pulmonary vein (PV), forming part of the intrinsic cardiac ganglia (red/black arrowheads). Note the vagal nerves surrounding the oesophagus (red arrow in A and black arrow in A’). B, B’ The ganglia stain positive for TUBB3 (red arrowheads). Nerve stainings are depicted separately in Supplemental Fig. 2. In mice (B) most ganglion cells still only express TUBB3 (yellow), although a few ganglion cells co-express ChAT (magenta) and even fewer TH (cyan). Vagal nerves are indicated by the red arrows. In humans (B’), the nerves remain undifferentiated (only TUBB3 expression). C, C’ Neural crest (-derived) cells at the dorsal mesenchymal protrusion between the PV clusters and the area of the developing atrioventricular (AV)-node (red arrow). D, D’ In the same area, small TUBB3+ nerves are present in both species, staining negative for TH and ChAT (red arrowheads).…

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  Fig. 8. NCCs and autonomic nerve markers in the right-sided SAN in mice and humans at mid-cardiogenesis. A–D Mouse sections of the right-sided SAN (blue/white dotted line) at E13.5. A’–D’ Human sections of the right-sided SAN (black/white dotted line) at 8WGA. A, A’ In both species, neural crest(-derived) cells (Wnt1Cre-GFP+ in mice and HNK-1+ in humans) are present at the right-sided SAN (red arrows). B, B’ In both species, TUBB3+ nerves are found in the SAN (red arrows and arrowheads). C-D, C’-D’ In mice, both parasympathetic [TUBB3+/ChAT+, red arrows in (B) and (C)] and sympathetic [TUBB3+/TH+, red arrowheads in (B) and (D)] nerves are present. Also note the ICA cells [TUBB3−/TH+, yellow arrowheads in (D)]. In human, the nerves remain undifferentiated [(red arrows in (B’)], evidenced by the negative staining for ChAT (C’) and TH (D’). Scalebars: 50 μm. RA: right atrium; RCV: right cardinal vein

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  Fig. 9. Autonomic nerve markers at the pulmonary vein in mice and humans at late cardiogenesis. A–D Mouse sections of the pulmonary vein at E18.5. The coronary sinus and dorsal right atrial wall with the orifice of the right superior caval vein and venous valves is also discernible. A’–D’ Human sections of the pulmonary vein at 13 WGA. A–D. In mouse embryos at this stage, most nerves at the venous pole co-express both ChAT and TH. A’–D’ Also in the human embryo of 13 WGA, many nerve cells co-express both ChAT and TH. Scalebars: 200 μm. Br: pulmonary bronchi; CS: coronary sinus; LA/RA: left/right atrium; LV/RV: left/right ventricle; Oe: oesophagus; PV: pulmonary vein

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  Fig. 10. Autonomic nerve markers at the right-sided SAN in mice and humans during late cardiogenesis. A–D Mouse sections of the right-sided SAN (white dotted line) at E18.5. Large TUBB3+ nerves are observed in the periphery of the SAN (white arrows), co-expressing both ChAT (C) and TH (D). Note the large amount of TH+/TUBB3− cells, i.e. ICA cells (B and C). A’–D’ Human sections of the right-sided SAN (white dotted line) at 11WGA. The SAN is highly innervated. Notably, the nerves are located more centrally compared to mice. ICA cells are not present. Scalebars: 100 μm. RCV; right cardinal vein