The heart’s little brain: shedding new light and CLARITY on the ‘black box’

Article, see (MS#318458) Hanna et al. “Innervation and Neuronal Control of the Mammalian Sinoatrial Node: A Comprehensive Atlas”

The field of neurocardiology is still in its infancy, yet its importance was recognised in 1628 by William Harvey when he noted: ‘For every affection of the mind that is attended with either pain or pleasure, hope or fear, is the cause of an agitation whose influence extends to the heart’. Harvey laid the foundation in the 17^th century for the neurologist Thomas Willis to describe the vagi as the ‘wandering nerves’ with cardiac projections. This observation guided Eduard and Heinrich Weber in the 1800s to make the first observation that right vagal stimulation slows heart rate. It also underpinned the 1936 Nobel Prize winning work of Otto Loewi and Henry Dale.

In 1921 Loewi established the chemical nature of neurotransmission. He demonstrated that stimulation of the right vagus nerve of an isolated frog heart decreased heart rate, and the transfer of the perfusate to a second frog heart produced bradycardia in the donor heart. The chemical substance in the perfusate responsible for this action was named ‘vagusstoff’ and was later identified as acetylcholine by Dale in the 1930s. Similar experiments to those by Loewi showed that the sympathetic nervous system also releases a chemical neurotransmitter ‘acceleranstoff’, subsequently identified as norepinephrine by Von Euler in 1946 with the development of sensitive catecholamine assays, leading to him being awarded the Nobel Prize for Physiology or Medicine in 1970 with Bernard Katz and Julius Axelrod.

The historical characterisation of the branches of the cardiac autonomic nervous system as simply an ‘accelerator and brake’ is broadly true, however underpinning this lies many layers of complexity¹. Functional studies demonstrate that there is a hierarchy of reflex responses occurring within the heart itself and the thoracic ganglia, which project all the way to the brainstem and higher centres. Communication between these anatomical levels is now becoming the textbook view². Even then, these hierarchical circuit diagrams, whilst a useful framework to model our understanding, in some ways detract from the complex neurocircuitry underneath.

The arrangement of afferent, efferent and interneurons and their architecture in ganglionic plexi in the epicardial fat pads was first referred to as the ‘heart’s little brain’ in the pioneering work of J Andrew Armour³. Immunohistochemistry has demonstrated a bewildering level of neuronal complexity, and despite these plexi remaining something of a ‘black box’, this has not stopped attempts at autonomic modulation therapies to prevent arrhythmogenesis associated with cardiac dysautonomia⁴. This is partly motivated by the relative accessibility of the ganglia and nerves to catheter ablation or electrical stimulation. However, these interventions have had limited success, which is perhaps due to the lack of precise anatomical targeting and a fundamental understanding of the biophysics underpinning this neurocircuitry.

Using cutting edge techniques that originate from neuroscience, such as tissue clearing/CLARITY, the article by Hanna et al⁵ in this issue of Circulation Research gives new anatomical and functional insight on the right atrial ganglionic plexus (RAGP) in the epicardial fat pad of the posterior wall of the right atrium. Whilst the same group has applied this technology to the mouse heart⁶, this is the first study to use both pig and human heart. This is a critical step and bridge to future translational studies, because rodent hearts lack the neural complexity within ganglionic plexi, express different co-transmitters compared to the human heart, and also present with a different physiological sympatho-vagal profile given patterns of innervation.

Of interest the RAGP contains glial cells, but predominately it is made up of cholinergic neurons (based on vesicular acetylcholine transporter, VAChT expression), many of which co-express neuropeptide-Y (NPY), thought originally only to be a sympathetic co-transmitter. However, functional release was not demonstrated. Calcitonin gene-related peptide (CGRP) and Substance P were also found in nerves tracking through the plexus only. Indeed, neurons expressing tyrosine hydroxylase (TH) did not seem to be typically adrenergic given the lack of expression of vesicular monoamine transporter 2 (VMAT2), although it may be that other isoforms are expressed. Some neurons also expressed both TH and VAChT, consistent with earlier reports showing switching of neurons between an adrenergic and cholinergic phenotype, which is well recognised following myocardial ischaemia⁷.

Nevertheless, a degree of caution is warranted when using only one or two markers to characterise a cell type. Emerging data from single cell RNA sequencing within the stellate ganglia⁸, has demonstrated that not every neuron expresses the full range of synthetic enzymes, pumps, and release channels that would make it an ‘adrenergic neuron’. To their credit Hanna et al. characterise neurons even further using high throughput screening PCR. Such work provides a more comprehensive atlas for researchers to delve into, and throws up surprises with regards to expression patterns and phenotypes.

The observations regarding NPY staining independent of TH in the RAGP, as well as there being a subset of TH positive neurons within the stellate ganglia that also express NPY⁸, is intriguing and deserves further study. Especially given the recent observations around the ability of NPY to induce cardiac arrhythmia⁹ and act as a biomarker for poor prognosis following both myocardial infarction⁹ and chronic severe heart failure¹⁰. Moreover, expression of muscarinic receptors and HCN channel transcripts within the RAGP and the stellate ganglia suggest that drugs like atropine and ivabradine may have more widespread effects beyond sinoatrial node cells themselves.

The paper is more than just a compelling ‘comprehensive atlas’ in terms of being descriptive or anatomical. The physiological observations are also significant. Bilateral (rather than just right) vagal inputs mediate control of sinoatrial node function in terms of leading pacemaker site and overall rate, AV node conduction and left ventricular contractility. The concept of parasympathetic control of the ventricles in large animals and humans was for many years controversial and only recently accepted to modulate both contractility¹¹ and susceptibility to ventricular arrhythmia independent of heart rate¹².

The authors also compare stimulation of the vagi with and without RAGP ablation, to direct stimulation of the RAGP itself. This highlights the importance of this plexus as a key hub not just in controlling sinoatrial node function, but also as a convergence point for vagal control of AV node and left ventricular function. It is noteworthy that transection of the vagi and bilateral stellate ganglionectomy referred to as ‘decentralisation’ enabled efferent vagal stimulation to induce a far more pronounced bradycardia, suggesting that neuronal excitability is modulated by these other influences and reflexes. The same careful and systematic approach taken in this work also needs to be applied to the other ganglionic plexi, in particular of the left atrium, together with exploration of the connections between them. It is encouraging to see that neuronal targets for intervention, can be delineated through discovery science and the approaches outlined here are a new foundation for this. It is also pleasing to see such an approach is being supported by the National Institute of Health’s SPARC (Stimulating Peripheral Activity to Relieve Conditions) program, which aims to accelerate development of therapeutic devices that modulate electrical activity in nerves to improve organ function. The potential to interact with the ‘heart’s little brain’ through direct stimulation, pharmacological or ablative approaches will increase as our understanding of the fundamental science deepens. In many ways we are still at the beginning of developing the framework in neurocardiology where target discovery can drive precise therapeutic interventions. Hanna and colleagues have provided important coordinates for this journey with their ‘comprehensive atlas’ roadmap.