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

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 (Ca 2+)-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 upand downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca 2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca 2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca 2+-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

Proarrhythmic Electrical Remodeling by Noncardiomyocytes at Interfaces With Cardiomyocytes Under Oxidative Stress

“…Like myofibroblasts, exogenous stem cells and progenitor cells (introduced by reparative/regenerative cellular or tissue therapies) express Cx43 and have more depolarized resting membrane potentials than native cardiomyocytes. For example, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have flourished as a powerful tool for drug discovery, disease modeling (Chow et al, 2013 ; Rocchetti et al, 2017 ; Chai et al, 2018 ; Wong et al, 2020 ), and regenerative medicine (Rhee and Wu, 2018 ; Sadahiro, 2019 ; Huang et al, 2020 ). However, in contrast to adult cardiomyocytes that have stable and more negative resting membrane potential (around −85 mV), hiPSC-CMs do not achieve sufficiently negative membrane potential (−40 to −70 mV), presumably due to inadequate expression of inward rectifier current I K1 (Goversen et al, 2018 ; Horváth et al, 2018 ).…”

Optimizing the Use of iPSC-CMs for Cardiac Regeneration in Animal Models

“…Consequently, several strategies have been employed to mature iPSC-CMs toward a more adult phenotype. Increased time in culture, physical and mechanical stimulation, engineered 3D or 4D constructs, hormonal stimulation and changing carbon substrates in culture media to modulate metabolism have all been shown to shift iPSC-CMs from a fetal to a more adult phenotype [37,40,45,70,101,113,[154][155][156]174,176,183,184,[216][217][218][219][220][221][222][223].…”

“…In addition to regulating the metabolism of fatty acids, it also upregulates the expression of the MHCα, the Na + /K + -ATPase, the Na + /Ca 2+ exchanger, voltage-gated potassium channels (Kv1.5, Kv4.2, Kv4.3) and the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) while downregulating the expression MHCβ and SERCA2-A regulator phospholamban (PLB). When applied to iPSC-CMs, the results comprised increased T-tubule development, faster calcium transient kinetics, faster AP kinetics, and an increase in maximum tension [169,176].…”

Using human induced pluripotent stem cell-derived cardiomyocytes to understand the mechanisms driving cardiomyocyte maturation

“…Moreover, T3 + electrical paced CMs cultured on substrate showed faster maximum upstroke velocity and increased I Na , lower automaticity as confirmed by reduced I f and HCN2/HCN4 expression, enhanced calcium handling demonstrated by gene expression and optical mapping calcium transients. RNA-seq analysis identified TGFB signaling as the key pathway downregulated in combinatorial treatments ( 138 ).…”