Project Summary/Abstract The recent increasing prevalence, severity, and healthcare burden of sinoatrial (SA) node dysfunction emphasize the need for more detailed studies of SA node functions that allow for effective therapy to treat and prevent SA node dysfunction. The major mechanisms of the dysfunction are the impaired ability of pacemaker cells to induce spontaneous rhythm (automaticity) and adverse remodeling in their electric conduction to surrounding atrial tissues (SA conduction). However, the current SA node or pacemaker models have been limited to theoretical models and isolated single cell-type cells or cell clusters, leaving a gap to model the autonomous cardiac contraction and heart rhythm and dysfunctions in automaticity and SA conduction. Moreover, the current single cell-type pacemakers worsened heart rhythm stability during one-month in vivo integration, which limits its application as a clinically viable biological pacemaker capable of generating robust pacemaking and conduction. To address the current limitation of SA node models, this proposal aims to develop a three-dimensional multicellular SA node organoid by reproducing human SA node’s multicellular tissue structure and fail-safe mechanisms. In contrast to the single cell-type biological pacemakers, human SA node is a natural organoid with elaborate insulated architecture and heterogeneous cellular composition. Moreover, the human SA node is equipped with redundant pacemaker sites and conduction pathways to protect the rhythm against adverse chronotropic stimulations. Thus, inspired by SA node’s structure and fail-safe mechanism, we aim at enhancing robustness in both automaticity and SA conduction: First, we will focus on enhancing automaticity of SA node organoids by identifying the expression of pacemaker membrane and calcium clock proteins, cell composition, and shape (Aim 1). Second, we will concentrate on improving conduction of SA node organoids by coordinating multiple pacemaker sites and conduction pathways (Aim 2). Last, we will evaluate the robustness of the SA node organoids in in vitro setting and in vivo atrioventricular block rodent model (Aim 3). These studies will define if tissue-level architecture and multicellular compositions mediate SA node’s robust pacemaking and conduction and may reveal a high-fidelity tissue-level biological pacemaker as a novel therapeutic strategy for SA node dysfunctions. The proposed organoids will be suitable for human preclinical testing assays to accelerate drug development, for dissecting patient-specific SA node disease pathophysiology, and for the development of implantable biological pacemakers.