Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10–12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70–90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

HCN channels underlie the depolarizing funny current (I_f) that contributes importantly to cardiac pacemaking. I_f is upregulated in failing and infarcted hearts, but its implication in disease mechanisms remained unresolved. We generated transgenic mice (HCN4^tg/wt) to assess functional consequences of HCN4 overexpression-mediated I_f increase in cardiomyocytes to levels observed in human heart failure. HCN4^tg/wt animals exhibit a dilated cardiomyopathy phenotype with increased cellular arrhythmogenicity but unchanged heart rate and conduction parameters. I_f augmentation induces a diastolic Na⁺ influx shifting the Na⁺/Ca²⁺ exchanger equilibrium towards ‘reverse mode’ leading to increased [Ca²⁺]_i. Changed Ca²⁺ homeostasis results in significantly higher systolic [Ca²⁺]_i transients and stimulates apoptosis. Pharmacological inhibition of I_f prevents the rise of [Ca²⁺]_i and protects from ventricular remodeling. Here we report that augmented myocardial I_f alters intracellular Ca²⁺ homeostasis leading to structural cardiac changes and increased arrhythmogenicity. Inhibition of myocardial I_fper se may constitute a therapeutic mechanism to prevent cardiomyopathy.