042-23 – Three-dimensional quantification of intrinsic neuronal networks in the heart reveals heterogeneous distribution of nerve fibres.

Background: The sinoatrial node (SAN) is the pacemaker of the heart and initiates the heartbeat. Control of SAN function by the autonomic nervous system allows rapid heart rate modulation to meet physiological demand. Consistently, 2D morphological investigations of SAN have shown enrichment of neurons. Here, we demonstrate a novel approach for 3D quantification of the intrinsic neuronal network using serial block face scanning electron microscopy (SBF-SEM).
Methods: SAN and atrial appendage biopsies (~1×1 mm) from male, C57BL/6J mice (n=3) were fixed, stained, and embedded in resin for electron microscopy. Serial images of blocks were acquired on FEI Quanta 250FEG SBF-SEM and electron micrograph stacks of the block face were manually analysed. Nerve fibres were annotated, and 3D reconstructions of neuronal networks performed using IMOD software. Data presented as mean±SD and P<0.05 was significant.
Results: SAN showed a heterogenous make-up composed of cardiomyocytes, blood vessels, fibroblasts, macrophages, and nerve fibres embedded in abundant collagen. Electron micrographs revealed numerous unmyelinated nerve fibres in SAN and few fibres were present in the atrial myocardium (AM). To investigate the extent of the intrinsic neuronal networks, 3D models of entire networks were constructed. SAN networks showed highly convoluted arrangement, with a high degree of convergence and divergence. AM networks exhibited a simple and wavy structure. The neuronal network volume was 19893±4499 µm3 in SAN vs. 1257±422 µm3 in AM (P=0.0182). Density of the neuronal network was 10.55±1.98 and 1.21±0.76 µm3 per 1000 µm3 of tissue block in SAN and AM (P=0.0016), respectively.
Conclusion: The SAN is densely innervated, and the interweaving architecture of its intrinsic neuronal network holds significance for pacemaker function and rapid chronotropic modulation. The 3D reconstructions provide the foundations for quantitative assessment of structural remodelling in SAN disease.