Poster T17 - Development Of A Multicellular Sinoatrial Node Organoid For Modeling Robust Pacemaking And Conduction

Introduction: The increasing prevalence of sinoatrial (SA) node dysfunction, driven by an aging population, is emerging as a significant health issue. The SA node, a natural pacemaker within the heart, consists of pacemaker cells embedded in a connective tissue matrix, electrically insulated from the surrounding atrial muscle cells. This isolated architecture, along with its heterogeneous cellular composition, is crucial for maintaining the SA node's robust pacemaking and electrical conduction. Developing methods to reconstruct this essential heterogeneous structure could offer a long-term solution for individuals suffering from SA node dysfunction. However, current biological pacemakers, which typically involve a single cell type, have shown to destabilize heart rhythm during one-month in vivo integration, posing a barrier to their clinical viability as long-term solutions.

Materials and

Methods: To address existing SA node model limitations, we developed a three-dimensional multicellular SA node organoid that reproduces the human SA node’s multicellular tissue structure and fail-safe mechanisms. First, inspired by the SA node’s partial electrical insulation, we structurally isolated a group of cardiomyocytes with a single exit pathway, allowing for electrical connection to neighboring cardiomyocytes. We investigated how this tissue-level architecture facilitates spontaneous cardiac rhythm production and overcomes source-sink mismatch with neighboring cardiomyocytes. Second, we overexpressed HCN4, a key pacemaker channel, in these isolated cardiomyocytes, further examining heart rhythm variability and the interplay between pacemaker gene expression and nodal architecture for long-term, high-fidelity pacing. Next, we developed an SA node organoid that replicates the human SA node's multicellular tissue structure. Initially, we established a protocol for generating cardiac SA node organoids by using a lentivirus to deliver doxycycline (dox)-inducible genes for key pacemaker transcription factors, specifically TBX-18 or Shox-2. We mixed wild-type human-induced pluripotent stem cells (hiPSCs) with those modified to express TBX-18 or Shox-2. These cell mixtures were aggregated, embedded in diluted Matrigel, and then subjected to a cardiac differentiation protocol, activating TBX-18 or Shox-2 at specific times (D1 or D7) via dox administration. Lastly, we integrated the SA node organoid into 2D atrial muscle tissue constructs, maintaining geometric isolation within the cardiac tissue to enhance source-sink mismatch.

Results, Conclusions, and Discussions: First, we found that the geometrically isolated cardiomyocytes successfully activated neighboring quiescent cardiomyocytes without any pacemaker-related gene expression, maintaining the rhythm for three months. Second, the overexpression of HCN4, a key pacemaker channel, in these isolated cardiomyocytes further improved heart rhythm variability down to the millisecond level, suggesting an interplay between pacemaker gene expression and nodal architecture for long-term, high-fidelity pacing. Third, hiPSC-based SA node organoids exhibited contractions lasting beyond 60 days, with notable upregulation of two transcription factors and pacemaker-specific genes following dox treatment. Finally, when we integrated the SA node organoid into 2D atrial muscle tissue constructs, the SA node organoids maintained geometric isolation within the cardiac tissue to enhance source-sink mismatch. This isolation allowed the SA node organoid to successfully trigger activity in adjacent dormant cardiomyocytes, preserving the rhythm for over 60 days.

These studies demonstrated that both tissue-level architecture and multicellular compositions mediated the SA node’s robust pacemaking and conduction. This approach may also reveal a high-fidelity, tissue-level biological pacemaker as a novel therapeutic strategy for SA node dysfunctions. Our SA node organoids will be suitable for human preclinical testing assays to accelerate drug development, dissect patient-specific SA node disease pathophysiology, and develop implantable biological pacemakers.

Acknowledgements (Optional): This research has been performed with the support of the American Heart Association Career Development Award #857583, a grant from National Heart Lung and Blood Institute 1R01HL161366-01A1, and cooperative research funds provided by SUMMIT Co., LTD, and the Korea Research Institute of Chemical Technology. S.-J.P. was funded by Georgia Institute of Technology, Emory University School of Medicine, and Emory School of Medicine Bridge