Tiered Sympathetic Control of Cardiac Function Revealed by Viral Tracing and Single Cell Transcriptome Profiling

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The cell bodies of postganglionic sympathetic neurons innervating the heart primarily reside in the stellate ganglion (SG), alongside neurons innervating other organs and tissues. Whether cardiac-innervating stellate ganglionic neurons (SGNs) exhibit diversity and distinction from those innervating other tissues is not known. To identify and resolve the transcriptomic profiles of SGNs innervating the heart we leveraged retrograde tracing techniques using adeno-associated virus (AAV) expressing fluorescent proteins (GFP or Td-tomato) with single cell RNA sequencing. We investigated electrophysiologic, morphologic, and physiologic roles for subsets of cardiac-specific neurons and found that three of five adrenergic SGN subtypes innervate the heart. These three subtypes stratify into two subpopulations; high (NA1a) and low (NA1b and NA1c) Npy-expressing cells, exhibit distinct morphological, neurochemical, and electrophysiologic characteristics. In physiologic studies in transgenic mouse models modulating NPY signaling, we identified differential control of cardiac responses by these two subpopulations to high and low stress states. These findings provide novel insights into the unique properties of neurons responsible for cardiac sympathetic regulation, with implications for novel strategies to target specific neuronal subtypes for sympathetic blockade in cardiac disease.

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  1. eLife assessment

    This important landmark paper identifies three distinct stellate ganglion nerve cell subtypes stratifiable in terms of their neuropeptide Y expression correlating these with gene expression and electrophysiological properties. Their innovative use of viral tracing techniques compellingly established their conclusions. This major contribution to cardiac sympathetic excitation is relevant to a wide scientific and clinical audience.

  2. Reviewer #1 (Public Review):

    The authors have achieved a demonstration of different stellate ganglion nerve cell functions and transmitter subtypes, of potential cardiac importance. They employ viral tracing techniques. These convincingly make this demonstration. The work will be key to our understanding of sympathetic function at the transmitter and physiological levels.

  3. Reviewer #2 (Public Review):

    The manuscript at hand by Sharma et al. presents new data on neurons of the stellate ganglia that are relevant for autonomic control of the heart. The authors identify stellate ganglionic neurons (SGN) that innervate the heart by retrograde tracing techniques and differentiate them from SGN neurons innervating other organs and tissues (mostly paw is used as a control). They subsequently employ single-cell RNAseq and morphological and functional (electrophysiological) studies. Their main finding is the identification of 3 SGN subtypes that they were further able to stratify into high and low neuropeptide Y cells. These subpopulations differ with regard to gene expression and action potential generation indicating different electrophysiological properties and different roles in the sympathoexcitation of the heart. They validate these findings by in vivo experiments where electrical stimulation of stellate ganglia after NPY-expressing neurons was depleted and find that heart rate change was lower under stimulation with high frequencies for NPY-depleted mice. The research question is very relevant and might have important therapeutic consequences for patients with cardiac diseases. The paper is written clearly. The methods applied are elegant and appropriate and the data support the conclusion.

    The authors do report on some experiments in which stellate ganglion was used. Viral administration and physiological studies were performed on the right, while RNA sequencing was done from the right and left stellate ganglion. As there are physiological lateral differences between the effects of the left and right stellate ganglion, it would be useful to thoroughly report which side was used for which experiment throughout the manuscript and to discuss whether any lateral differences are relevant for the obtained results and conclusions.

  4. Reviewer #3 (Public Review):

    Using viral tracing and single-cell transcriptome profiling the authors investigated the electrophysiologic, morphologic, and physiologic roles for subsets of cardiac-specific neurons and found evidence that three adrenergic stellate ganglionic neuron subtypes innervate the heart.

    The presented findings provide relevant insights into the properties of neurons modulating cardiac sympathetic control. The findings might open up new avenues to targeted modulation of cardiac sympathetic control. Additional insights from various models addressing for example ischemic and non-ischemic cardiomyopathy might allow to development of targeted therapies for various patient populations in the future.