PhD student Rostock University Medical Center Rostock, Mecklenburg-Vorpommern, Germany
Abstract: This study characterizes the differentiated sinoatrial node (SAN) microenvironment using induced pluripotent stem cells (iPSCs) enhanced by adenoviral vector (AdV)-mediated transcription factor (TF) delivery. By mimicking the SAN’s complex cellular and molecular landscape, we aim to provide insights into its function and structure. Using AdV to overexpress key TFs - Tbx18, Shox2 and Isl1 - we directed iPSCs to differentiate into SAN-like cells. Proteomics and electrophysiology studies, together with publicly available single-cell RNA sequencing were used to assess and compare extracellular matrix components, ion channels and TFs critical for SAN development. Differentiated SAN cells exhibited distinct electrophysiological properties, including shorter action potential durations and higher contraction frequencies than ventricular cardiomyocytes. Immunocytochemical staining confirmed the presence of SAN-specific subpopulations. Additionally, fibroblasts and extracellular matrix proteins such as collagen III and IV supported the structural and functional integrity of the SAN microenvironment. The pacemaker-like properties of SAN-derived cells were validated by assessing the effect of ivabradine (IVA) on cycle length. SAN cells displayed intrinsic pacemaker activity with shorter cycle lengths than ventricular myocytes (VMs). IVA treatment significantly prolonged the cycle length of SAN cells, confirming If current inhibition, while minimally affecting VMs. The complete protocol showed robust pacemaker activity, while intermediate responses in the SAN-Tbx18 group highlighted subpopulation diversity and potential immaturity. This study highlights the critical role of the SAN microenvironment in maintaining pacemaker activity and establishes AdV-mediated TF expression as an efficient approach for generating functionally distinct SAN cells. These findings advance regenerative medicine strategies targeting cardiac dysfunction and provide a powerful platform for exploring cardiac biology.
