Introduction
Neuronal control of ventricular myocardium has traditionally been considered to be exerted by the sympathetic branch of the intracardiac nervous system (ICNS), while effects of parasympathetic neurons were thought to be restricted to atrial tissue. However, multiple clinical studies reported that impaired parasympathetic neuronal activity correlated with an increased susceptibility to ventricular arrhythmias1. In addition, research using rodent models revealed the presence of parasympathetic innervation of the ventricles2. In this project, we aim to systematically characterize the structure of parasympathetic neurons projecting to the ventricles and their effects on ventricular function, to better understand the integration of seemingly opposing ICNS signals.
Methods
We use mouse models expressing the light-gated cation channel Channelrhodopsin-2 (ChR2-eYFP) in cholinergic (ChAT-ChR2) or adrenergic neurons (TH1-ChR2). For structural analysis, hearts were excised and tissue cleared using an optimized X-CLARITY protocol, in combination with antibody staining of the branch of the ICNS not expressing eYFP, and of pre- and post-junctional receptor proteins. Processed tissue was imaged in 3D using confocal microscopy. In order to optogenetically or electrically stimulate ICNS neurons, we have established an adapted Langendorff-perfusion system. This is based on cannulation of the descending rather than the ascending aorta, and ensures preservation of the extracardiac sympathetic ganglia and their projections to the heart (Fig. 1A).
Results and outlook
We show that sympathetic and parasympathetic axons run next to one-other in murine ventricles (Fig. 1B) and frequently feature synaptic varicosities. We observe an abundance of muscarinic receptors expressed at the surface of ventricular cardiomyocytes. In first functional experiments, we find that optogenetic activation of ChAT-ChR2 neurons decreases heart rate, delays AV-node conduction and induces ventricular arrhythmias (Fig. 1C), in line with our previous study applying different concentrations of carbachol3. Experiments with optogenetic stimulation of sympathetic neurons are on-going. In the future, we plan to co-activate the two ICNS branches using optogenetics and either electrical or pharmacological stimulation, to disentangle ICNS control and signal integration between its two branches, in respect to ventricular electrophysiology (action potential duration, conduction velocity, arrhythmogenesis). With this research, we aim to re-assess textbook knowledge that largely neglects the control of ventricular activity by intracardiac cholinergic neurons, an important prerequisite to developing therapeutic interventions targeting the ICNS, ultimately aiming to prevent ventricular arrhythmias.
1. Brack KE et al. Mechanisms underlying the autonomic modulation of ventricular fibrillation initiation—tentative prophylactic properties of vagus nerve stimulation on malignant arrhythmias in heart failure. Heart Fail Rev. 2013;18:389-408.
2. Rajendran PS et al. Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies. Nat Commun. 2019;10:1944.
3. Sassu E et al. Age-related structural and functional changes of the intracardiac nervous system. J Mol Cell Cardiol. 2024;187:1-14.
Fig. 1: Experimental setup (A), structural (B) and functional (C) pilot data (ECG of optogenetically stimulated ChAT-ChR2 heart at two frequencies).
