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.
Highlights
ATP-sensitive potassium (KATP) channels, located on the sarcolemma and mitochondrial inner membranes of mature CMs, play an important role in cardioprotection during conditions such as ischemia by linking membrane excitability to metabolism[12,13,14,15,16,17,18]
To exploit the potential use of T3 for driven tissue maturation, we examined the functional consequences of their treatments on a multi-cellular 3D ventricular cardiac microtissue system, where true dynamic tension developed by the tissue in real time, rather than shortening of single hESC-CMs or their clusters as an surrogate index for contractile forces[35,36], can be measured
Neither T3 treatment nor electrical conditioning affected sarcIK, ATP; this mirrored the upregulation of mito IK, ATP by T3 but not hypoxia, indicating cue-specific effects on sarc and mito IK, ATP
Summary
ATP-sensitive potassium (KATP) channels, located on the sarcolemma and mitochondrial inner membranes of mature CMs, play an important role in cardioprotection during conditions such as ischemia by linking membrane excitability to metabolism[12,13,14,15,16,17,18]. When cytoplasmic ATP is depleted (e.g., during ischemia)[20,21], sarcKATP opens to allow a repolarizing outward K+current, IK, ATP , thereby shortening the action potential (AP) and subsequently lowering Ca2+ influx to create a negative inotropic effect for metabolic conservation[14,22,23,24]. Despite the crucial physiological role of KATP channels and the promises of hESC-CMs, neither sarc nor mitoIK, ATP in hESC-CMs has ever been studied. The present study aims to fill this gap, while seeking biological insights and developing effective means for driving maturation of hESC-CMs
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