Introduction
Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, with a strong genetic component that contributes to the disease. The coordinated contraction of atrial muscle depends on regulatory networks, in which transcription factors drive downstream effectors to maintain proper cardiac conduction. The transcription factor SHOX2 is involved in the development and function of the sinoatrial node (SAN), the primary pacemaker of the heart, and is one of the main pacemaker genes associated with conduction-related diseases. SHOX2 variants have been identified in early-onset AF and sinus node dysfunction patients and functionally characterised in different model systems.

Method
To model SHOX2-related conduction dysfunction in a human system, iPSCs have been generated from AF patients carrying a functional non-coding SHOX2 variant in the 3´UTR (c.*28T/C; rs138912749) and a SHOX2 coding variant (c.849C>A), together with isogenic control lines. We differentiated these cells to nodal and atrial cardiomyocytes and established a workflow to perform functional and transcriptional analyses.

Results
We isolated SHOX2 positive iPSC-derived nodal cardiomyocytes from a mixed population. The workflow included a differentiation and maturation period, sorting of cardiomyocytes from heterogenous cell pools, labelling of SHOX2 positive cells via viral transduction of lineage-specific promoter-driven fluorescent reporter gene constructs, and subsequent FACS sorting. Single-cell RNA sequencing of SHOX2 pacemaker cells was performed to dissect transcriptional profiles of patient- and isogenic control lines, to assign specific alterations and uncover drivers of dysfunctional signalling pathways. We showed that SHOX2 expression and function are significantly impaired in patient-derived nodal and atrial cells compared with isogenic control cells. Furthermore, action potential characteristics and expression of genes involved in cardiac electrophysiology and development were significantly altered in patient-derived cells. We could also show that miR-92b-5p is able to bind to the 3’UTR variant and thus has the potential to have a regulatory effect on SHOX2 expression, providing a mechanistic understanding of this disease.

Discussion
Our data show that this human iPSC model of SHOX2-related AF reflects both the phenotype seen in early-onset AF patients and Shox2 animal models, and is well suited to understand how SHOX2 variants influence the occurrence and progression of atrial arrhythmias.