Functional Expression and Regulation of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels (HCN) in Mouse iPS Cell-derived Cardiomyocytes after UTF1-Neo Selection

Abstract

Background/Aims: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. Methods: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. Results: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and If currents as well as functional hormonal regulation and sensitivity to If channel blockers. Conclusion: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed If current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes.

Keywords

• Cardiomyocytes
• Heart development
• Induced pluripotent stem cells
• Hyperpolarization-activated cation channel
• Whole-cell patch-clamp