The sinoatrial node (SAN) initiates and regulates the heartbeat in mammals. Sinoatrial (SA) nodal cells generate spontaneous action potentials by the combination of Ca++ oscillatory mechanisms and the activation of membrane ion channels, including the hyperpolarization activated cyclic nucleotide-gated channels (HCN). HCN4 is the most abundant subunit in the SAN and mediates the inward depolarizing current If. If has been suggested as isolation mechanism of the SAN to resist the hyperpolarizing force of the surrounding atrium. HCN4 relevance to pacing has been further supported by the discovery of more than twenty different mutations associated with mild or severe forms of arrhythmias. When SA nodal cells are damaged by aging or disease, the implantation of an electronic pacemaker is required. Electronic devices have been improved for decades but they still exhibit inadequate autonomic response, limited battery life, and risk of lead corrosion and infection, often causing adverse cardiac remodeling. These limitations could be addressed by the generation of biological cell-based pacemakers. Although different strategies have been proposed to develop biological pacemakers, to date, clinically relevant studies using human cells have not been carried out yet. In this study, we propose a cell-based gene therapy approach, where the non-viral integrating transposon system Sleeping Beauty (SB) is used to overexpress hHCN4 in induced pluripotent stem cells (hiPSCs) as a platform for differentiation of pacemaker cardiomyocytes. I have developed a complete workflow for the generation of hHCN4-overexpressing cardiomyocytes (hHCN4 hiPSC CM) that includes reprogramming of human fibroblasts into hiPSCs, hHCN4 overexpression in hiPSCs by SB-mediated gene delivery, and optimal cardiomyocyte differentiation into hHCN4 hiPSC CM. hHCN4-overexpressing hiPSCs showed stable, long-term expression of If while maintaining pluripotency. These cells efficiently differentiated into cardiomyocytes showing comparable yields to control hiPSCs. hHCN4 hiPSC CM cultures displayed robust beating and contained 80% of cells expressing cardiomyocyte-specific markers from day 9 onwards through cardiomyocyte differentiation. Compared to cardiomyocytes derived from control hiPSC lines, hHCN4 hiPSC CM showed 3-10 fold larger If that, just like in pacemaker cells, activated slowly at -60mV, was blocked by Cs+ and was susceptible to ß-adrenergic regulation. Electrophysiological and transcriptional comparison of control and hHCN4 hiPSC CM showed equivalent action potential properties and similar expression profile of ion channels, connexins and genes involved in general cardiac function or specific to SAN function and development. The number of spontaneously beating cells was similar, being this automaticity independent of the level of expression of If in both control and hHCN4 hiPSC CM. However, spontaneously beating hHCN4 hiPSC CM displayed faster beating frequency and were enriched in cells with pacemaker-like morphology. Further in vitro and in vivo studies, including co culture with ventricular-like cardiomyocyte monolayers and transplantation into the heart of a large animal model, are needed to determine the ability of hHCN4 hiPSC CM to couple electrically and pace cardiac tissue and ultimately, the heart.
