Moreover, the vast majority of data regarding the development of the SAN is derived from animal models ( van Eif et al., 2018) and our insights into human SAN development are very limited ( Csepe et al., 2016 Chandler et al., 2009 Sizarov et al., 2011 van Eif et al., 2019). While the shared origin of pacemaker cells with proepicardium and further differentiation of the pacemaker cells to distinct subpopulations is recognized, the mechanisms underlying these processes are poorly understood. Furthermore, transitional cells (SAN-TZ) with transcriptional and functional properties intermediate to that of pacemaker cells and the adjacent atrial myocardium have also been reported, which are believed to play a critical role in transmitting the electrical impulses from the SAN to the adjacent atrial myocardium ( Boyett et al., 2000 Csepe et al., 2016 Goodyer et al., 2019 Li et al., 2019). The SAN-tail located inferior to the SAN-head expresses Tbx3 and Nkx2-5 but is devoid of Tbx18 ( Wiese et al., 2009 Goodyer et al., 2019). This region is also distinct from all other cardiomyocytes in the heart as it lacks the expression of Nkx2-5 ( Wiese et al., 2009).

The pacemaker cells in the SAN-head population express the T-box transcription factors Tbx18 and Tbx3. Within the cardiomyocyte fraction of the SAN, there are distinct subpopulations such as head, tail, and transitional cells ( Komosa et al., 2021). In vitro studies using human pluripotent stem cells point to a crosstalk between BMP, retinoic acid (RA) and wingless-related integration site (WNT) signaling in the differentiation of pacemaker and proepicardial cells ( Wiesinger et al., 2021). In chicken development, bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling have been shown to orchestrate the separation of myocardial and proepicardial cells ( Kruithof et al., 2006 van Wijk et al., 2009). Moreover, proepicardium-derived mesenchymal cells have been shown to be integral for remodeling and sustained electrical activity of the SAN ( Bressan et al., 2018). The pacemaker cells of the SAN originate from a Tbx18 + progenitor population that also gives rise to proepicardial cells ( van Wijk et al., 2009 Mommersteeg et al., 2010). The mechanisms that regulate the development of the various cell types of the SAN niche remain largely unknown. Previous studies have revealed that the SAN is a complex heterogeneous structure composed of both myocardial (head, tail, transitional cells) and non-myocardial cells such as fibroblasts, smooth muscle cells, etc., which contribute to its function ( Wiese et al., 2009 Bressan et al., 2018 Goodyer et al., 2019). A better understanding of the origin, composition, and function of the human SAN will enable the development of effective therapies.

Dysfunction of the SAN results in potentially life-threatening bradyarrhythmia ( Choudhury et al., 2015) and current treatment with the implantation of electronic pacemakers is suboptimal ( Cingolani et al., 2018 Boink et al., 2015). Each heartbeat is triggered by the electrical impulses generated by the sinoatrial node (SAN), referred to as the primary pacemaker of the heart. The human heart beats about 3 billion times in an average life span. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN. Furthermore, we uncovered roles for TGFβ and WNT signaling in the branching of transitional and head SANCM subtypes, respectively.

Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into ‘transitional’, ‘tail’, and ‘head’ subtypes. Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart.

Any human heart title sequence