Influence of Electromechanical Activity on Cardiac Differentiation of Mouse Embryonic Stem Cells

During differentiation, mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) receive electromechanical cues from spontaneous beating. Therefore, promoting electromechanical activity via electrical pacing or suppressing it by drug treatment might affect the cellular functional development. Electrical pacing was applied to confluent monolayers of mESC-CMs during late-stage differentiation (days 16-18). Alternatively, spontaneous contraction was suppressed by (a) blocking ion currents with CsCl (HCN channel), trazodone (T-type Ca²⁺ channel), or both CsCl and trazodone on days 11-18; or (b) applying blebbistatin (excitation-contraction uncoupler) on days 11-14. Electrophysiological properties and gene expression were examined on day 19 and 18, respectively. Optical mapping revealed no significant difference in conduction velocity (CV)in paced vs. non-pacedmonolayers, nor were there significant changes in gene expression of connexin-43, Na-Ca exchanger (NCX), or myosin heavy chain (MHC). However, CV variability among differentiation batches and CV heterogeneity within individual monolayers were significantly lower in paced mESC-CMs. Alternatively, while the four drug treatments suppressed contraction with varying degrees (up to complete inhibition), there was no significant difference in CV for any of the treatments compared with controls. Trazodone treatment significantly reduced CV variability as compared to controls, whereas CsCl treatment significantly reduced CV heterogeneity. Distinct changes in gene expression of connexin-43, MHC, HCNl, Cav3.1/3.2 were not observed. Electrical pacing, but not suppression of spontaneous contraction, during late-stage differentiation reduces the intrinsic variability of CV among differentiation batches and across individual monolayers, which can be beneficial in the application of ESCs for myocardial tissue repair.