Triiodothyronine and dexamethasone alter potassium channel expression and promote electrophysiological maturation of human-induced pluripotent stem cell-derived cardiomyocytes
Graphical abstract
Introduction
The human-induced pluripotent stem cell (hiPSC) technology opens new avenues for human disease modeling and drug screening. HiPSC-derived cardiomyocytes (hiPSC-CMs) can recapitulate the structure and function of adult cardiomyocytes (CMs): (1) express cardiac-specific markers, sarcomere proteins, and calcium handling regulators [[1], [2], [3]], and (2) express multiple cardiac ion channels, thus generating a typical human action potential (AP) [2,3]. Unfortunately, hiPSC-CMs are structurally and functionally immature, resembling fetal CMs [4]. This immature phenotype of hiPSC-CMs affects their usability. Therefore, there is an urgent need to mature hiPSC-CMs.
HiPSC-CMs poorly recapitulate electrophysiological properties of adult human cardiomyocytes (CMs). In adult CMs, three K+ currents tightly regulate the configuration of AP: the rapid (IKr) and slow (IKs) delayed rectifier K+ currents and the inward rectifier current (IK1). Highly expressed IK1, IKr and IKs ensure stable and constant AP signaling in adult CMs. The hyperpolarization-activated, “funny” current (If) is a mixed Na+ and K+ inward current activating at voltages below −40/−45 mV [5]. If can trigger spontaneous APs and generate pacemaking activity [5,6]. In the adult heart, If only exists in pacemaker cells dedicated to generating spontaneous activity and modulating sympathetic acceleration capacities of the heart rate [5]. Unfortunately, hiPSC-CMs typically have a large amount of If but a low amount or near-complete absence of IK1, IKr and IKs [7]. These features reduce the utility of hiPSC-CMs for disease modeling and drug screening [8]. Therefore, increased IK1, IKr and IKs and reduced If would be crucial to improving the fidelity of hiPSC-CMs as human CM models.
How can we improve the electrophysiological maturation of hiPSC-CMs? In humans, fetal CMs require around 280 days to reach a neonatal phenotype. The heart's electrophysiological maturation process involves upregulating IK1, IKr, and IKs, and reducing If in ventricle [9,10]. However, hiPSC-CMs are normally generated in the dish within 30 days [1], and they resemble the first trimester of the human fetal heart (gestational weeks 7–10) [4]. Unsurprisingly, day 30th hiPSC-CMs typically exhibit low IK1, IKr, and IKs but high If. Hence, one logical way to achieve a more mature phenotype is to increase the culture time of hiPSC-CMs. Sartiani et al. [11] demonstrated that culture durations greater than 3 months increased IK1 and IKr and reduced If in human embryonic stem cell derived-CMs (hESC-CMs). Wang et al. [12] also found that culturing more than 80 days can shorten APD and increase conduction velocity (CV) in hESC-CM monolayers. Recently, Feyen et al. [13] used physiological levels of glucose and calcium and a mixture of fatty acids to culture hiPSC-CMs over 21 to 35 days, which significantly increased IK1. Therefore, the critical challenge is how to increase IK1, IKr, and IKs and reduce If in hiPSC-CMs rapidly and consistently.
More evidence suggests the significant roles of triiodothyronine (T3) and dexamethasone (Dex) in the CM maturation. Several reports have documented that T3 and Dex can upregulate transcription levels for K+ channels in CMs, respectively [[14], [15], [16], [17]]. Dex has also been reported to facilitate the K+ channel trafficking via serine/threonine protein kinase (SGK) and enhance their membrane expression [[18], [19], [20]]. Our previous study showed the combined T3 and Dex can promote the functional T-tubule development in hiPSC-CMs [21]. However, the effects of T3 + Dex on the K+-channel expression in hiPSC-CMs were not examined. In this study, we treated hiPSC-CMs with a combination of T3 + Dex for 14 days. We found that T3 + Dex treatment changed the gene expression of K+ channels, increased IK1, IKr, and IKs, and reduced If, thereby enhancing the electrophysiological maturation of hiPSC-CMs.
Section snippets
Methods
HiPSC lines were approved by Vanderbilt University Medical Center Institutional Review Board.
Conduction velocity and action potential duration in hiPSC-CM cell sheets
We used optical mapping to measure CV and APD at 80% repolarization (APD80) in FluVolt-loaded hiPSC-derived cardiomyocyte cell sheets (hiPSC-CCSs) at different electrically-stimulated pacing-cycle lengths (400, 500, 666, 800, and 1000 ms). Fig. 1A shows examples of optical AP signals derived from vehicle- and T3 + Dex-treated hiPSC-CCSs when paced at a cycle length of 1000 ms. Fig. 1B and C depict the resulting APD80 and CV restitution curves obtained from the vehicle- and T3 + Dex- treated
Discussion
This study reports the effects of T3 and Dex on the electrophysiological properties of hiPSC-CMs. We found that T3 + Dex treatment: (1) increased CV and shortened APD80 in hiPSC-CCs; (2) slowed spontaneous beating rate of single hiPSC-CMs; (3) led to a more hyperpolarized RMP, faster Vmax, and shorter APD in single hiPSC-CMs; (4) changed the expression of genes encoding for K+ channels; (5) increased INa, IK1, IKr, and IKs, and reduced If in hiPSC-CMs.
Conclusion
T3 + Dex treatment increased CV and shortened APD in hiPSC-CCSs. Moreover, in single hiPSC-CMs, T3 + Dex treatment slowed spontaneous beating rates and led to a more hyperpolarized RMP, faster Vmax, and shorter APD. These changes were mediated by increasing INa, IK1, IKr, and IKs, and reduced If in T3 + Dex-treated hiPSC-CMs. Thus, our results demonstrate that T3 + Dex treatment is a practical approach to improve electrophysiological maturation of hiPSC-CMs within 30 days of cardiac
Disclosure of funding
The work was supported by Leducq Foundation 18CVD05 (to B.C.K. and L.G.), by the National Institutes of Health NHLBI R35 HL144980 (to B.C.K.), by the European Research Council ERC-2017-COG-773181-iPS-ChOp-AF (L.G.), by American Heart Association 19POST34380182 (to L.L.W.), by Heart Rhythm Society Clinical Research Award in Honor of Mark Josephson and Hein Wellens (to Y.W.), by National Institutes of Health NHLBI T32 HL007411-39 (to J.S.). There are no relationships with the industry. The
References (54)
- et al.
Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes
J. Mol. Cell. Cardiol.
(2018) - et al.
Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories
J. Mol. Cell. Cardiol.
(2015) - et al.
Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes
Cell Rep.
(2020) - et al.
Effects of thyroid and glucocorticoid hormones on Kv1.5 potassium channel gene expression in the rat left ventricle
Biochem. Biophys. Res. Commun.
(1997) - et al.
Thyroid hormone regulates mRNA expression and currents of ion channels in rat atrium
Biochem. Biophys. Res. Commun.
(2003) - et al.
Dexamethasone rapidly induces Kv1.5 K+ channel gene transcription and expression in clonal pituitary cells
Neuron
(1993) - et al.
The serum- and glucocorticoid-inducible kinases SGK1 and SGK3 regulate hERG channel expression via ubiquitin ligase Nedd4-2 and GTPase Rab11
J. Biol. Chem.
(2013) - et al.
Patient-independent human induced pluripotent stem cell model: a new tool for rapid determination of genetic variant pathogenicity in long QT syndrome
Heart Rhythm.
(2019) - et al.
Human induced pluripotent stem cell-derived cardiac cell sheets expressing genetically encoded voltage Indicator for pharmacological and arrhythmia studies
Stem Cell Rep.
(2018) - et al.
Dexamethasone and stress upregulate Kv1.5 K+ channel gene expression in rat ventricular myocytes
Neuropharmacology
(1996)
Phenotype-based high-throughput classification of long QT syndrome subtypes using human induced pluripotent stem cells
Stem Cell Rep.
Amiodarone reduces transmural heterogeneity of repolarization in the human heart
J. Am. Coll. Cardiol.
Matrigel mattress: a method for the generation of single contracting human-induced pluripotent stem cell-derived cardiomyocytes
Circ. Res.
Transcriptome of human foetal heart compared with cardiomyocytes from pluripotent stem cells
Development
The role of the funny current in pacemaker activity
Circ. Res.
Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity
J. Cardiovasc. Electrophysiol.
High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents
Am. J. Physiol. Heart Circ. Physiol.
Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias
Front. Physiol.
Large-scale analysis of ion channel gene expression in the mouse heart during perinatal development
Physiol. Genomics
Developmental changes in ionic channel activity in the embryonic murine heart
Circ. Res.
Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: a molecular and electrophysiological approach
Stem Cells
Contribution of potassium channels to action potential repolarization of human embryonic stem cell-derived cardiomyocytes
Br. J. Pharmacol.
Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells
Mol. Endocrinol.
Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome
FASEB J.
Regulation of endocytic recycling of KCNQ1/KCNE1 potassium channels
Circ. Res.
Thyroid and glucocorticoid hormones promote functional T-tubule development in human-induced pluripotent stem cell derived cardiomyocytes
Circ. Res.
Chemically defined generation of human cardiomyocytes
Nat. Methods
Cited by (12)
Cardiac maturation
2024, Journal of Molecular and Cellular CardiologyMultifactorial approaches to enhance maturation of human iPSC-derived cardiomyocytes
2023, Journal of Molecular LiquidsPLK inhibitors identified by high content phenotypic screening promote maturation of human PSC-derived cardiomyocytes
2022, Biochemical and Biophysical Research CommunicationsCitation Excerpt :PCM1 is localized around γ-tubulin+ centrosome region at day 11, then gradually translocates to perinuclear region at day 18 (Fig. 1H), suggesting that PCM1 localization at the NE in hPSC-CMs can be used as a quantifiable marker for hPSC-CM maturation. Combination of Triiodothyronine (T3) and Dexamethasone (Dex) have been reported to promote cardiomyocyte maturation [20,21]. To further determine if PCM1 is correlated with hPSC-CM maturation and select a robust positive control for the HCS assay, we tested the effect of T3/Dex on hPSC-CM maturation and PCM1 localization.
Deciphering the roles of triiodothyronine (T3) and thyroid-stimulating hormone (TSH) on cardiac electrical remodeling in clinical and experimental hypothyroidism
2024, Journal of Physiology and Biochemistry