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Background Parasympathetic control of the heart is an important component in the regulation of normal cardiac function. However, the anatomic course of parasympathetic innervation of the heart is unclear. Objective The purpose of this study was to apply a gross parasympathetic nerve stain technique to reveal the details of the morphology of the cardiac parasympathetic nervous system. Methods Ten whole pig hearts were stained using a histochemical method. Immediately after sacrifice, hearts were placed in a buffered solution containing acetylthiocholine, which precipitates with acetylcholinesterase, allowing identification of cholinergic nerves. The epicardial and endocardial surfaces of the atria and ventricles were examined for nerve thickness and density. Results In both atria, nerve density was significantly greater on the endocardium, but nerve thickness was significantly greater on the epicardium. The right atrium (RA) was more densely innervated than the left atrium (LA) on the endocardium, whereas the LA was more densely innervated than the RA on the epicardium. In the ventricles, numerous thick cholinergic nerves were clearly identifiable across the epicardium, generally running parallel to the left anterior descending artery. The endocardial surfaces of the ventricles revealed a dense network of fine parasympathetic nerve fibers. As in the atria, nerve density was greater on the ventricular endocardium, but nerve thickness was greater on the epicardium. The right ventricle (RV) was more densely innervated than the left ventricle (LV), whereas the LV endocardium was more densely innervated than the RV endocardium. Conclusion The epicardial and endocardial surfaces of the atria and ventricles are richly innervated by parasympathetic nerves. The density of parasympathetic innervation is heterogeneous across both the epicardial and endocardial surfaces of the heart.

The parasympathetic (P) nervous system is thought to contribute significantly to focal atrial fibrillation (AF). Thus we hypothesized that P nerve fibers [and related muscarinic (M2) receptors] are preferentially located in the posterior left atrium (PLA) and that selective cholinergic blockade in the PLA can be successfully performed to alter vagal AF substrate. The PLA, pulmonary veins (PVs), and left atrial appendage (LAA) from six dogs were immunostained for sympathetic (S) nerves, P nerves, and M2 receptors. Epicardial electrophysiological mapping was performed in seven additional dogs. The PLA was the most richly innervated, with nerve bundles containing P and S fibers (0.9 ± 1, 3.2 ± 2.5, and 0.17 ± 0.3/cm2 in the PV, PLA, and LAA, respectively, P < 0.001); nerve bundles were located in fibrofatty tissue as well as in surrounding myocardium. P fibers predominated over S fibers within bundles (P-to-S ratio = 4.4, 7.2, and 5.8 in PV, PLA, and LAA, respectively). M2 distribution was also most pronounced in the PLA (17.8 ± 8.3, 14.3 ± 7.3, and 14.5 ± 8 M2-stained cells/cm2 in the PLA, PV, and LAA, respectively, P = 0.012). Left cervical vagal stimulation (VS) caused significant effective refractory period shortening in all regions, with easily inducible AF. Topical application of 1% tropicamide to the PLA significantly attenuated VS-induced effective refractory period shortening in the PLA, PV, and LAA and decreased AF inducibility by 92% ( P < 0.001). We conclude that 1) P fibers and M2 receptors are preferentially located in the PLA, suggesting an important role for this region in creation of vagal AF substrate and 2) targeted P blockade in the PLA is feasible and results in attenuation of vagal responses in the entire left atrium and, consequently, a change in AF substrate.

The objective of the study was to investigate the morphology, distribution, and electrophysiological profile of the autonomic fibers that innervate the ligament of Marshall (LOM). Gross anatomical dissections were performed in 10 dogs. Sections of the left vagus nerve, left stellate ganglion, and the LOM were immunostained to identify adrenergic and cholinergic nerves. Hearts were also stained for acetylcholinesterase to identify epicardial cholinergic nerves. In vivo electropyhsiological studies were performed in another 10 dogs before and after LOM ablation. The anatomical examination revealed that the LOM is innervated by a branch of the left vagus. Immunohistochemistry confirmed that these nerve bundles are predominantly cholinergic (cholinergic-to-adrenergic ratio of 12.6 ± 3.9:1). Cholinergic nerves originating in the LOM were found to innervate surrounding left atrial structures, including the pulmonary veins, left atrial appendage, coronary sinus, and posterior left atrial fat pad. Ablation of the LOM significantly attenuated effective refractory period shortening at distant sites, such as pulmonary veins and left atrial appendage, in response to vagal stimulation (vagal-induced ERP decrease in the left atrium: baseline vs. postablation = 17 vs. 4%; P = 0.0056). In conclusion, the LOM contains a predominance of cholinergic nerve fibers. Cholinergic fibers arising from the LOM innervate surrounding structures and contribute to the electrophysiological profile of the left atrium. These findings may provide a basis for the role of the LOM in the genesis and maintenance of atrial fibrillation.