Nerves projecting from the intrinsic cardiac ganglia of the pulmonary veins modulate sinoatrial node pacemaker function

Abstract

Aims: Pulmonary vein ganglia (PVG) are targets for atrial fibrillation ablation. However, the functional relevance of PVG to the normal heart rhythm remains unclear. Our aim was to investigate whether PVG can modulate sinoatrial node (SAN) function.

Methods and results: Forty-nine C57BL and seven Connexin40+/EGFP mice were studied. We used tyrosine-hydroxylase (TH) and choline-acetyltransferase immunofluorescence labelling to characterize adrenergic and cholinergic neural elements. PVG projected postganglionic nerves to the SAN, which entered the SAN as an extensive, mesh-like neural network. PVG neurones were adrenergic, cholinergic, and biphenotypic. Histochemical characterization of two human embryonic hearts showed similarities between mouse and human neuroanatomy: direct neural communications between PVG and SAN. In Langendorff perfused mouse hearts, PVG were stimulated using 200-2000 ms trains of pulses (300 μs, 400 µA, 200 Hz). PVG stimulation caused an initial heart rate (HR) slowing (36 ± 9%) followed by acceleration. PVG stimulation in the presence of propranolol caused HR slowing (43 ± 13%) that was sustained over 20 beats. PVG stimulation with atropine progressively increased HR. Time-course effects were enhanced with 1000 and 2000 ms trains (P < 0.05 vs. 200 ms). In optical mapping, PVG stimulation shifted the origin of SAN discharges. In five paroxysmal AF patients undergoing pulmonary vein ablation, application of radiofrequency energy to the PVG area during sinus rhythm produced a decrease in HR similar to that observed in isolated mouse hearts.

Conclusion: PVG have functional and anatomical biphenotypic characteristics. They can have significant effects on the electrophysiological control of the SAN.

Keywords: Intrinsic cardiac ganglia; Pulmonary veins; Sinoatrial node.

Figure 1

Neuroanatomical characterization of the mouse whole-mount atrial preparation and PVG (A–C). Arrowheads indicate extrinsic nerves accessing the mouse heart, whereas double arrowheads point to intrinsic ganglia located at the roots of PVs. Arrows indicate nerves emerging from the PVG, which extend to the SAN region. Boxed areas are enlarged in Supplementary material online, Figure S1. (D–F) Characterization of the PVG (green, TH; red, ChAT). LCV, left cranial vein; LPV, left pulmonary vein; MPV, middle pulmonary vein; RPV, right pulmonary vein; CS, coronary sinus.

Figure 2

Immediate effects (average of three P–P intervals post-stimulation) of PVG stimulation on HR. (A) PVG stimulation (n = 12); RAA stimulation (n = 23); *P < 0.05 vs. pre-stimulation. (B) PVG stimulation + propranolol (n = 17); RAA stimulation + propranolol (n = 24); *P < 0.05 vs. pre-stimulation. (C) PVG stimulation + atropine (n = 13); RAA stimulation + atropine (n = 16); *P < 0.05 vs. pre-stimulation. (D) PVG stimulation atropine + propranolol (n = 16); RAA stimulation atropine + propranolol (n = 16); (E) *P < 0.05 vs. PVG stimulation + atropine and PVG stimulation atropine + propranolol; ^#P < 0.05 vs. 200 ms train.

Figure 3

Time-course effects (20 beats after stimulation) of PVG stimulation with (A) 200 ms train, (B) 1000 ms train, and (C) 2000 ms train. PVG stimulation (n = 12); PVG stimulation + propranolol (n = 17); PVG stimulation + atropine (n = 12); PVG stimulation atropine + propranolol (n = 16).

Figure 4

Modulation of the SAN activation pattern induced by PVG stimulation. The leading pacemaker site shifted upwards (A) in 31.8% of observations (seven of 22), after applying 1000 ms train of stimulation to the PVG. (B) A representative example of a downward shift after PVG stimulation. This took place in 68.2% of observations (15 of 22). (C) The increase in cycle length after 1000 ms trains. The quantification of the shift in the site of earliest activation is in (D). N = 8 hearts, n = 32 observations. SVC, superior vena cava; PV, pulmonary veins; RAA, right atrial appendage; arrows point towards the tip of the stimulation electrode. Colour bars in (A) and (B) display the activation time in milliseconds.

Figure 5

Combined EGFP fluorescence microscopy and optical mapping in isolated atrial preparation from Cx40^+/EGFP mice. (A) PVG stimulation produced an increase in the P–P interval (P < 0.05 vs. pre-stimulation, top of the panel). Representative traces of a single pixel from the SAN area on (C) are shown before (black) and after stimulation (red). (B) EGFP fluorescence image of the SAN superimposed on the optical image of the atrial preparation. The white arrow points to the SAN artery. Activation maps showing the site of earliest activation with respect to the SAN artery before (C) and after PVG stimulation (D). Colour bars in (C) and (D) display the activation time in milliseconds.

Figure 6

(A) Histochemical characterization of human embryonic PV-LA and SAN whole-mount preparations. Ganglia located at the roots of the PVs project nerve fibres (white arrows) to the SAN area. (B) Evolution of the P–P interval during control and the first 12 intervals following RFA to the PV roots during sinus rhythm (n = 5). Carto maps showing the sites of RFA at the roots of the left superior pulmonary vein (C, LSPV) and the right superior pulmonary vein (D, RIPV). Red arrows indicate the sites at which RFA induced slowing. (E) ECG tracings illustrate the slowing of HR during RFA at the root of the LSPV (top) and the RIPV (bottom). RF current was applied around the PV antrum, power <35 W (<25 W over the posterior LA), temperature <43°C.