Problem
Despite substantial advances in understanding the anatomy and physiology of cardiac nerves that contribute to therapeutic responses, we are still in need of a better understanding of the structural organization of cardiac innervation to effectively use specific neuromodulation treatment instead of non-specific treatments for cardiovascular diseases. The neuroanatomical organization of cardiac sympathetic innervation is very complex and remains poorly understood. Unraveling the comprehensive topographical map of the catecholaminergic efferent axons in the heart could give us important insights into the architecture of cardiac sympathetic nerves and provide the foundation for future functional studies of sympathetic control of the heart.
Solution
Inspired and supported by SPARC, we mapped the connections of the peripheral nerves (sympathetic) and the heart, and reconstructed the whole axonal network in the atria using tools (Neurolucida 360, MBF Bioscience) developed by other SPARC-supported groups to accomplish this common goal.
To look at the cardiac nerve architecture, flat-mounts of the whole right and left atria and ventricles were immunolabeled for tyrosine hydroxylase (TH, a sympathetic marker). We then utilized confocal microscopy to image the whole specimen and Neurolucida 360 software to trace, digitize, and quantitatively map the topographical distribution of the cardiac sympathetic postganglionic innervation.
Figure 1: Top panel: Distribution of TH-IR axons in the flat-mount of whole left and right atria (connected). Bottom panel: Digitized tracing of TH-IR bundles, each bundle is represented by a different color.
A montage of several hundred maximal projection images showed several large bundles which bifurcated into smaller bundles which innervated the entire atria (Figure 1, top panel). In the atria, a few large TH-immunoreactive (IR) axon bundles entered both atria, branched into small bundles and then single axons that eventually formed very dense terminal networks in the epicardium, myocardium and inlet regions of major vessels to the atria (right:SVC, IVC, and LPCV; left: pulmonary veins). Varicose TH-IR axons formed close contact with cardiomyocytes, vessels, and adipocytes. Multiple intrinsic cardiac ganglia (ICG) were identified in the epicardium of both atria, and a subpopulation of the neurons in the ICG were TH-IR. Most TH-IR axons in bundles traveled through ICG before forming dense varicose terminal networks in cardiomyocytes. We did not observe varicose TH-IR terminals encircling ICG neurons.
TH-IR neurons in the intrinsic cardiac ganglia (ICG) were more densely distributed in the left atrium. TH-IR axons were observed running through the ICG, innervating blood vessels, muscles, and fat tissue.
Neurolucida 360 tracing and digitizing of the TH-IR bundles and axons revealed 4 major bundles entered the atria mainly at the superior vena cava (SVC), left pre-caval vein (LPCV), and left atrial-pulmonary vein junction then bifurcated into small bundles that eventually ramified into individual varicose axons (Figure 1, bottom panel). Two bundles extended TH-IR axons toward the right atrium while the other two bundles mainly projected toward the left atrium. These four bundles innervated distinct regions with a certain degree of overlap.
The Impact
This work contributes to our overall understanding of the cardiac-sympathetic brain connectome. It provides an anatomical foundation for functional mapping of sympathetic control for the heart, as well as for the evaluation of the remodeling of cardiac sympathetic innervation in chronic disease models (including obstructive sleep apnea, hypertension, heart failure). This mapping could be utilized to understand the sympathetic specific control of different regions of the atria and their autonomic responses, information which could lead to specific treatments for cardiovascular diseases.
And finally, beyond the heart, the approach used in this study can be used as an atlas for future studies and can be applied to map the sympathetic innervation in other organs in both normal and diseased models.
