Establishment of hyperpolarization-activated cyclic nucleotide-gated potassium channel 4-overexpressing mouse embryonic stem cell-derived cardiomyocytes as a candidate for a biological pacemaker

Abstract

Purpose: The number of patients receiving cardiac pacemaker implantation has been increasing. However, the devices have various problems including short battery life, lead breaks, infection, and electromagnetic interference. Alternative therapies including cell therapy are expected to solve these problems. Cell therapies and gene therapies to establish biological pacemakers have been reported, but a physiological heart rate has never been achieved. Therefore, we generated hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)-overexpressing mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) and investigated whether they have characteristics as a functional pacemaker in vitro. Methods: A pEF1-rabbit Hcn4-IRES-ZsGreen1 vector was used for nucleofection of mouse embryonic stem cells (mESCs) and selection of stable clones that are G418-resistant and ZsGreen1-positive. The mESCs were differentiated to cardiomyocytes by the hanging drop method. The mESC-CMs were purified under serum/glucose-free and lactate-supplemented conditions. The characteristics of the purified cells were verifid by RT-PCR and immunostaining, and the frequency of spontaneous beating was compared between control and HCN4-overexpressing mESC-CMs. Results: Most of the purified cells showed spontaneous beating and they were alpha-cardiac actinin-positive by immunostaining. RT-PCR showed that Oct4 and Nanog had disappeared and that ion channels (Hcn4, Scn5a, Connexin40, Connexin43, and Connexin45), Nkx2.5 and Tnnt2 were positive. HCN4-overexpressing mESC-CMs expressed higher levels of Hcn4 than did control mESC-CMs. HCN4-overexpressing mESC-CMs showed significantly more rapid beating than did control mESC-CMs (Hcn4-overexpressing, 87.4±11.9 /min; control, 43.1±4.8 /min). The beating rate of HCN4-overexpressing mESC-CMs decreased in response to ivabradine (no treatment, 105±18.9 /min; 3 μM ivabradine, 74.0±14.7 /min; 30 μM ivabradine, 37.5±4.1 /min) and increased in response to isoproterenol (no treatment, 93.0±8.9 /min; 1 μM isoproterenol, 124±6.5 /min). Conclusions: We established HCN4-overexpresssing mESC-CMs that show rapid spontaneous beating and responses to drugs in vitro. The results indicate the possibility of application of these cells as a biological pacemaker.