Abstract The sinus node initiates pacemaking activity in mammalian hearts with robust regularity. Sinus node dysfunction is a common, heterogenous disorder resulting in syncope, fatigue, and chronotropic incompetence. No pharmacologic therapy current exists for sinus node dysfunction, and the only available therapeutic modality is implantation of a permanent pacemaker. Although the canonical hierarchy of pacemaking activity states that atrioventricular foci lead pacemaking activity when the sinus node fails, experimental data suggests that subsidiary right atrial pacemakers (SAPs) emerge, mature, and lead pacemaking function in the absence of the sinus node. The intrinsic cardiac nervous system (ICNS), a network of discrete ganglia distributed on the epicardial surface of the heart, modulates regional cardiac electrical and mechanical activity on a beat-by-beat basis. Dysfunction of the ICNS has been implicated in various rhythm disorders including sinus node dysfunction, though clinical interventions targeting the ICNS have reported mixed results. The right atrial ganglionated plexus (RAGP) and inferior vena cava-inferior atrial ganglionated plexus (IVC-IA GP) robustly innervate the rostral and caudal right atrium, where SAPs typically emerge. These SAPs are influenced by the autonomic nervous system, and disruption of autonomic innervation may impact the maturation and function of these SAPs. In this proposal, we aim to evaluate the role of the ICNS in the maturation of SAPs in a porcine model following excision of the sinus node. In Aim 1, we will evaluate the emergence of SAPs using continuous ECG- telemetry, in vivo electrophysiologic mapping, and autonomic challenges before and after excision of the sinus node. We will then characterize atrial and pacemaker protein and gene expression profiles using immunohistochemistry and RNA-sequencing in the SAP compared to sinus node and remote atrium. In Aim 2, we will selectively remove the RAGP or IVC-IA GP and excise the sinus node, and evaluate the emergence of SAPs using similar techniques. This study will determine the role of the ICNS in establishing pacemaker function, and elucidate whether subsidiary pacemaking function is dependent on neuronal input. In addition, structural and transcriptomic characteristics of SAPs will be defined, providing insight into pathways that mediate pacemaking independent of the sinus node complex.