SPARC: Autonomic Innervation of Porcine Ventricular Myocardium and Purkinje Fibers
Authors: Hoover Donald B. , Hanna Peter , Dacey Michael J. , Hadaya Joseph E. , Swid Mohammed A. , Smith Elizabeth H. , Peirce Stanley G. , Poston Megan D. , Potter Jacqueline C. , Ardell Jeffrey L. , Shivkumar Kalyanam 
Published: April 2020

Previous studies demonstrated that porcine ventricular myocardium receives abundant innervation, but most of this work focused on nerves at epicardial and endocardial surfaces. Furthermore, our knowledge regarding cholinergic innervation of ventricular muscle remains sparse. The goal of this study was to identify and quantify the regional abundance of cholinergic and noradrenergic nerves in left and right ventricular walls from the base of the heart to the apex. One‐year old male and female Yucatan minipigs were sacrificed, hearts were removed, and blood was flushed from the tissue by infusing heparinized saline into the coronary arteries. Samples of myocardium were collected from regions of interest, and these were immersion‐fixed overnight in cold 4% paraformaldehyde/0.2% picric acid in buffer before cryoprotection. Transmural 30 μm sections were cut in a cryostat and collected on slides for labeling of nerve fibers by fluorescence immunohistochemistry. Adjacent sections were stained for the pan‐neuronal marker protein gene product 9.5 (PGP9.5) or double‐labeled for vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH) to identify cholinergic and noradrenergic nerves, respectively. Digital images of labeled sections were collected and deconvoluted using an Olympus BX41 microscope with a 20X objective, DP74 camera, and cellSense Dimension software. Nerve densities were determined as % area using ImageJ software. Abundant labeling for all markers was detected across the ventricular walls, and nerve densities were comparable at all levels from base to apex. Total nerve density based on PGP9.5 staining ranged from ~2.5 to 4 % area in the left ventricles, and the sum of TH and VAChT nerve densities accounted for most of the total. For the majority of sections on the left, TH nerves were somewhat more abundant than VAChT nerves, but average cholinergic nerve density was at least 1% area. The ratio of noradrenergic/cholinergic nerves was ~1.5 to 2. Preliminary evidence indicates cholinergic and noradrenergic nerves have similar densities in right ventricle. Staining of sections for the glial cell marker S100 showed that Schwann cells were present throughout the ventricles. Purkinje fibers were identified at the endocardium of many sections, and these cells stained intensely for PGP9.5 but did not stain for another neuronal marker microtubule associated protein 2. The Purkinje cells received abundant cholinergic innervation with single nerves occasionally penetrating fiber bundles and wrapping around Purkinje cells. In contrast, noradrenergic innervation of Purkinje fibers was sparse. Our findings provide definitive evidence for prominent and ubiquitous innervation of porcine ventricular muscle by both noradrenergic and cholinergic nerves, providing a basis for balanced regulation of contractile function. In marked contrast, cholinergic innervation is more abundant than noradrenergic in the Purkinje system, suggesting a dominant role of parasympathetic nerves in controlling impulse propagation via the cardiac conducting system.Support or Funding InformationSupported by NIH 3OT2OD023848