Abstract Body: The rising prevalence of sinoatrial (SA) node dysfunction, attributed to the aging population, is emerging as a critical health issue. The SA node, a natural organoid within the heart, comprises the pacemaker cells embedded within a connective tissue matrix, insulated electrically from the surrounding atrial muscle cells. This isolated architecture, along with a heterogeneous cellular composition, is crucial for the SA node's robust pacemaking and electrical conduction. Developing methods to reconstruct this crucial heterogeneous structure could offer a long-term solution for individuals with SA node dysfunction. However, the use of current biological pacemakers, which typically involve a single cell type, has often led to unstable heart rhythms during one-month in vivo integration, challenging their long-term clinical application as biological pacemakers.

To address existing SA node model limitations, we developed a SA node organoid that reproduces the human SA node's multicellular tissue structure. Initially, we developed a protocol for generating cardiac SA node organoids by employing a lentivirus to deliver 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 a diluted Matrigel, and then exposed to a cardiac differentiation protocol, activating TBX-18 or Shox-2 at specific times (D1 or D7) via Dox administration. By D9, these organoids began to exhibit contraction, which lasted beyond 60 days, with notable upregulation of two transcription factors and pacemaker-specific genes following Dox treatment. Subsequently, we integrated the SA node organoid into 2D hiPSC-derived atrial muscle tissue constructs, maintaining 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 findings could redefine the role of tissue architecture and cell diversity in the SA node's pacemaking function, potentially leading to a high-fidelity, tissue-level biological pacemaker as a new treatment strategy for SA node dysfunctions.