Non-cell autonomous cues for enhanced functionality of human embryonic stem cell-derived cardiomyocytes via maturation of sarcolemmal and mitochondrial KATP channels

Abstract

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs), but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs, sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study, we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK, ATP in single hESC-VCMs was dormant under baseline conditions, but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ~8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly, sarcIK, ATP displayed a ~3-fold increase after treatment with hypoxia (5% O2). MitoIK, ATP was absent in hESC-VCMs. However, the thyroid hormone T3 up-regulated mitoIK, ATP, conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system, T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK, ATP.

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

“…the downregulation of their molecular correlates HCN2 and HCN4. As for Ca 2+ handling, which is of central importance to excitation-contraction coupling, such related gene products as PLN and SERCA were upregulated, leading to improved calcium handling and subsequent contractility (Keung et al, 2016;Li et al, 2019). Other groups have reported similar findings where electrical pacing in hiPSC-CM tissue constructs promoted contractile force by ∼20-fold along with ∼2-and ∼1.5-fold increases in protein expression of SERCA and RYR, respectively, when compared to untreated controls (Ruan et al, 2016) or sensitivity toward isoproterenol, as indicated by calcium imaging (Ronaldson-Bouchard et al, 2019).…”

“…Over the past decade or so, significant progress has been made in ventricular (V) specification to obtain high-purity VCMs (Ebert et al, 2012;Spater et al, 2014;Weng et al, 2014) for cardiac tissue engineering (Tallawi et al, 2015;Kolanowski et al, 2017;Wong et al, 2019). However, 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). Electrophysiologically, hPSC-CMs spontaneously fire action potentials (AP) with slower kinetics, starkly contrasting their quiescent-yet-excitable adult counterparts (Lieu et al, 2013).…”

Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells

“…Most of the designs are based on specific tissue-engineered formats using methods such as SU-8 photolithography 22,23 , bioprinting 24,25 , decellularization of whole hearts 26 , surface micropatterning [27][28][29] , biowires 23,30 , or casting molds as hydrogel (collagen, Matrigel or fibrinogens) scaffolds 22,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] . Engineered heart tissues (EHTs) are grown on mechanical supports or pillars providing mechanical load and have been described to induce hiPSC-CM maturation 35,37 .…”

“…One system uses forced supraphysiological pacing up to 6 Hz to facilitate this 40 . The initial designs 48 were low-throughput, time-consuming and costly for both academic laboratories and pharma but miniaturized versions have now been made 22,30,34,36,42,45,47 .…”

Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications

“…The platform can be adapted to single cells, anisotropic sheets, and tissue strips. Their human ventricular cardiac organoid chambers provide a comprehensive bioreactor model enabling force and electrophysiology measurements with minimal manipulation [ 130 ].…”