Topographical Distribution and Morphology of CGRP-IR Axons in the Flat-Mounts of Whole Mouse and Rat Hearts.

To understand and treat cardiac pain, it is important to have an understanding of the topographical distribution of nociceptive axons in the heart. While previous studies have detected nociceptive axons in the sectioned heart, their full distribution has not yet been determined. In this study, we used a nociceptive marker calcitonin gene-related peptide (CGRP) to determine the distribution of nociceptive afferent axons in the whole heart. We first removed the hearts of male Sprague-Dawley rats (n=6), and C57BL/6 mice (n=6). The hearts were dissected into left and right atria and ventricles and interventricular septum, and prepared as flat-mounts. These samples were then processed with immunohistochemical labeling using a primary antibody which binds to CGRP and an Alexa Fluro 488 secondary antibody for fluorescence. The flat-mounts were imaged with a Zeiss M2 Imager (automatic fluorescence microscope) using a 488 nm excitation wavelength. In both rats and mice, we found that CGRP-IR nerve bundles entered the heart and innervated the left and right atria and ventricles. For the right/left atria, CGRP-IR axons entered as large bundles near the superior/inferior vena cava, left pre-caval vein and the pulmonary veins before bifurcating into small branches, and finally formed single varicose axons and terminals which distributed throughout the tissue, including cardiac ganglia, the SA/AV nodes, auricles, and the blood vessels. In the right/left ventricles and interventricular septum, CGRP-IR axons entered as large bundles through the base before bifurcating into small branches towards the apex Ultimately, single varicose axons and terminals innervated myocardium and blood vessels. Our data shows the distribution of CGRP-IR axons in the whole heart at single cell/axon resolution. With this information, we will gain insight into the function of these axons and the pathways that they follow, paving the way for the development of better treatments for cardiac pain and disease. The first two authors contributed equally to this work.