Enhancement of pacing function by HCN4 overexpression in human pluripotent stem cell-derived cardiomyocytes

Abstract

Background The number of patients with bradyarrhythmia and the number of patients with cardiac pacemakers are increasing with the aging population and the increase in the number of patients with heart diseases. Some patients in whom a cardiac pacemaker has been implanted experience problems such as pacemaker infection and inconvenience due to electromagnetic interference. We have reported that overexpression of HCN channels producing a pacemaker current in mouse embryonic stem cell-derived cardiomyocytes showed enhanced pacing function in vitro and in vivo. The aim of this study was to determine whether HCN4 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can strengthen the pacing function of the cells. Methods Human HCN4 was transduced in the AAVS1 locus of human induced pluripotent stem cells by nucleofection and HCN4-overexpressing iPSC-CMs were generated. Gene expression profiles, frequencies of spontaneous contraction and pacing abilities of HCN4-overexpressing and non-overexpressing iPSC-CMs in vitro were compared. Results HCN4-overexpressing iPSC-CMs showed higher spontaneous contraction rates than those of non-overexpressing iPSC-CMs. They responded to an HCN channel blocker and β adrenergic stimulation. The pacing rates against parent iPSC line-derived cardiomyocytes were also higher in HCN4-overexpressing iPSC-CMs than in non-overexpressing iPSC-CMs. Conclusions Overexpression of HCN4 showed enhancement of If current, spontaneous firing and pacing function in iPSC-CMs. These data suggest this transgenic cell line may be useful as a cardiac pacemaker.

Effect of mechanical stretching stimulation on maturation of human iPS cell-derived cardiomyocytes co-cultured with human gingival fibroblasts

“…Immunofluorescence staining of iPS-CMs results showed that cTnT-positive cells (red) and HCN4-positive cells (green) were observed in both groups, and they were high expressed in the stretch group compared with the control group. HCN4 channels are required for normal rhythm generation and conduction, playing a key role in cardiac pacing 21 , and HCN4 overexpression shows enhanced I(F) current, spontaneous discharge, and pacing in iPS-CMs 22 , which is consistent with our finding of increased cardiomyocytes contractility in the stretch group (Figure 5B). Experiments also confirmed the existence of sarcomere structure in the stretch group, these results indicating the maturation of myocardial tissue.…”

Conversion of Unmodified Stem Cells to Pacemaker Cells by Overexpression of Key Developmental Genes

“…During induced conversion, most cells adopt the spindle shape. HCN2 expression was able to induce the ASC conversion in culture, similar to previously tested HCN2 expression in MSCs following transplantation to the sites of SAN damage [17,20] or in cultured cardiac-like cells [24,25,33,37]. SHOX2 alone was not efficient in converting ASCs into differentiating cells [23,26].…”

“…Previously, TBX5 was part of the recipes used for cell transdifferentiation into cardiomyocytes [45][46][47] and it likely stimulated NKX2.5 expression. In the case of ASCs, TBX5 expression potentially contributes to their cardiac differentiation making them somewhat closer to NKX2.5-expressing cardiac and cardiac-like cells used by other researchers [21,24,25,29,35,37,38,[41][42][43][44]. Furthermore, TBX5, which positively regulates the levels of SHOX2 and TBX3 in the developing may induce the expression of these pacemaker lineage genes during ASC conversion.…”

In vivo tracking transplanted cardiomyocytes derived from human induced pluripotent stem cells using nuclear medicine imaging

“…“pAAVS1_SA_loxP_PGK_ Neo _loxP/CAG_IRES_ EGFP/AmpR ” that we previously generated was used for a donor vector ( 15 ). Human SLC5A5 open reading frame (ORF) subcloned into pCMV6-XL5 vector (Cat.#.…”