Abstract Body: Alterations of bioelectrical signals at early embryonic stages may cause heterotaxia. It has been
shown that the lack of the pacemaker channel HCN4 at the very early stage of Xenopus laevis
development induces heterotaxia and malformed hearts. This project aims to better characterize
these non-canonical roles of HCN4 channels during the early phase of mouse embryonic stem
cells (mESC) differentiation into Embryoid Bodies (mEBs).
Two experimental approaches were adopted: 1) generation of mESC HCN4 knock out (KO) lines
through CRISPR-Cas9 technique and 2) chronic pharmacological inhibition of the pacemaker
channel (by ivabradine, Iva). mESC were differentiated through the hanging drop technique.
Molecular and functional evaluations were performed throughout the first 8 days of mESC
differentiation. Changes in the time-dependent expression of the three germ layer markers
(Brachyury-T, GATA4 and Nodal), pluripotency markers (Oct-4 and Nanog) and cardiac markers
(i.e. TnI) were evaluated. Cardiac differentiation was functionally evaluated by monitoring the
spontaneous mEB beating occurrence, furtherly characterized through a Multi-Electrode Array
system associated to a laser-based intracellular reading mode.
To generate HCN4 KO mESC lines (1^st approach), we designed three 20-nt length sgRNAs and
two of them were selected based on results of the genomic analysis. As expected, time dependent
increase of HCN4 transcript levels were observed during the first 8 days of mESC differentiation
(CTR group), but it was abolished in both HCN4 KO lines. In comparison to CTR, in both KO lines,
HCN1, GATA-4 and TnI transcript levels were less expressed at each differentiation time point; in
contrast, Oct4 and Nanog levels were more expressed in KO lines, suggesting a crucial role of
HCN4 in the early phase of the mESC differentiation process. To note, HCN4-KO dependent
effects on mESC differentiation were largely reproduced by pacemaker channel blockade by Iva
(3-10 µM, 2^nd approach). In particular, in comparison to untreated cells, Iva-treated cells showed
lower HCN4, HCN1, TnI, GATA-4 levels, and higher Oct-4 levels.
To summarize, HCN4 misregulation largely affects the mESC differentiation process, suggesting a
functional role of the pacemaker channel on the early cardiac development; this further supports
the idea that bioelectricity is fundamental for a proper cellular development.