Combinatorial Treatment of Human Cardiac Engineered Tissues With Biomimetic Cues Induces Functional Maturation as Revealed by Optical Mapping of Action Potentials and Calcium Transients.

Andy On-Tik Wong,Chi-Wing Kong,Hongkai Wu,Yiu-Fai Cheung,Michelle Khine,Nicodemus Wong,Wendy Keung,Ronald A Li,Maggie Zi-Ying Chow,Lin Geng,Kevin D Costa,Eugene K Lee

doi:10.3389/fphys.2020.00165

Abstract

Although biomimetic stimuli, such as microgroove-induced alignment (μ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined μ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-β signaling was downregulated; the TGF-β receptor agonist and antagonist TGF-β1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.

Highlights

The self-renewing property of human pluripotent stem cells, including induced pluripotent stem cells and embryonic stem cells (ESC), offers a potentially unlimited supply of cardiomyocytes (CMs)
The anisotropic ratio (AR) for control was 1.0, which was consistent with the radial conduction of action potentials (AP) from the point of origin, as shown in the isochronal map
Representative immunostaining images of α-actinin in flat, 8, 10, and 15 μ groups of human ventricular cardiac anisotropic sheets (hvCAS) were used to demonstrate the change in tissue morphology (Supplementary Figure 1)

Summary

Introduction

The self-renewing property of human pluripotent stem cells (hPSC), including induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC), offers a potentially unlimited supply of cardiomyocytes (CMs). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) possess immature fetal-like properties (Lieu et al, 2013; Robertson et al, 2013; Poon et al, 2015; Keung et al, 2016; Kadota et al, 2017; Ronaldson-Bouchard et al, 2019). Conduction is non-anisotropic and prone to arrhythmias unless the constituent cells are aligned (Chen et al, 2011; Luna et al, 2011; Wang et al, 2013; Shum et al, 2017). Immature calcium handling properties (Liu et al, 2007, 2009; Li et al, 2013; Chen et al, 2015) contribute to weaker contractile functions (Ruan et al, 2016)

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