scholarly journals Large-Scale Simulation of the Phenotypical Variability Induced by Loss-of-Function Long QT Mutations in Human Induced Pluripotent Stem Cell Cardiomyocytes

2018 ◽  
Vol 19 (11) ◽  
pp. 3583 ◽  
Author(s):  
Michelangelo Paci ◽  
Simona Casini ◽  
Milena Bellin ◽  
Jari Hyttinen ◽  
Stefano Severi

Loss-of-function long QT (LQT) mutations inducing LQT1 and LQT2 syndromes have been successfully translated to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) used as disease-specific models. However, their in vitro investigation mainly relies on experiments using small numbers of cells. This is especially critical when working with cells as heterogeneous as hiPSC-CMs. We aim (i) to investigate in silico the ionic mechanisms underlying LQT1 and LQT2 hiPSC-CM phenotypic variability, and (ii) to enable massive in silico drug tests on mutant hiPSC-CMs. We combined (i) data of control and mutant slow and rapid delayed rectifying K+ currents, IKr and IKs respectively, (ii) a recent in silico hiPSC-CM model, and (iii) the population of models paradigm to generate control and mutant populations for LQT1 and LQT2 cardiomyocytes. Our four populations contain from 1008 to 3584 models. In line with the experimental in vitro data, mutant in silico hiPSC-CMs showed prolonged action potential (AP) duration (LQT1: +14%, LQT2: +39%) and large electrophysiological variability. Finally, the mutant populations were split into normal-like hiPSC-CMs (with action potential duration similar to control) and at risk hiPSC-CMs (with clearly prolonged action potential duration). At risk mutant hiPSC-CMs carried higher expression of L-type Ca2+, lower expression of IKr and increased sensitivity to quinidine as compared to mutant normal-like hiPSC-CMs, resulting in AP abnormalities. In conclusion, we were able to reproduce the two most common LQT syndromes with large-scale simulations, which enable investigating biophysical mechanisms difficult to assess in vitro, e.g., how variations of ion current expressions in a physiological range can impact on AP properties of mutant hiPSC-CMs.

2019 ◽  
Author(s):  
M Paci ◽  
E Passini ◽  
A Klimas ◽  
S Severi ◽  
J Hyttinen ◽  
...  

AbstractHigh-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) technology and by contact-free all-optical systems simultaneously measuring action potential (AP) and Ca2+ transient (CaTr). Parallel computational advances have shown that in silico models can predict drug effects with high accuracy. In this work, we combine these in vitro and in silico technologies and demonstrate the utility of high-throughput experimental data to refine in silico hiPS-CM populations, and to predict and explain drug action mechanisms. Optically-obtained hiPS-CM AP and CaTr were used from spontaneous activity and under pacing in control and drug conditions at multiple doses.An updated version of the Paci2018 model was developed to refine the description of hiPS-CM spontaneous electrical activity; a population of in silico hiPS-CMs was constructed and calibrated using the optically-recorded AP and CaTr. We tested five drugs (astemizole, dofetilide, ibutilide, bepridil and diltiazem), and compared simulations against in vitro optical recordings.Our simulations showed that physiologically-accurate population of models can be obtained by integrating AP and CaTr control records. Thus constructed population of models predicted correctly the drug effects and occurrence of adverse episodes, even though the population was optimized only based on control data and in vitro drug testing data were not deployed during its calibration. Furthermore, the in silico investigation yielded mechanistic insights, e.g. through simulations, bepridil’s more pro-arrhythmic action in adult cardiomyocytes compared to hiPS-CMs could be traced to the different expression of ion currents in the two.Therefore, our work: i) supports the utility of all-optical electrophysiology in providing high-content data to refine experimentally-calibrated populations of in silico hiPS-CMs, ii) offers insights into certain limitations when translating results obtained in hiPS-CMs to humans and shows the strength of combining high-throughput in vitro and population in silico approaches.SignificanceWe demonstrate the integration of human in silico drug trials and optically-recorded simultaneous action potential and calcium transient data from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) for prediction and mechanistic investigations of drug action. We propose a population of in silico models i) based on a new hiPS-CM model recapitulating the mechanisms underlying hiPS-CM automaticity and ii) calibrated with all-optical measurements. We used our in silico population to predict and evaluate the effects of 5 drugs and the underlying biophysical mechanisms, obtaining results in agreement with our experiments and one independent dataset. This work supports the use of high-content, high-quality all-optical electrophysiology data to develop, calibrate and validate computer models of hiPS-CM for in silico drug trials.


EP Europace ◽  
2020 ◽  
Vol 22 (9) ◽  
pp. 1431-1441
Author(s):  
Cristina Altrocchi ◽  
Tessa de Korte ◽  
Joyce Bernardi ◽  
Roel L H M G Spätjens ◽  
Stefan R Braam ◽  
...  

Abstract Aims Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. Methods and results Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation–contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. Conclusion Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Keisuke Sekine ◽  
Shimpei Ogawa ◽  
Syusaku Tsuzuki ◽  
Tatsuya Kobayashi ◽  
Kazuki Ikeda ◽  
...  

Abstract Advances in organoid technology have broadened the number of target diseases and conditions in which human induced pluripotent stem cell (iPSC)-based regenerative medicine can be applied; however, mass production of organoids and the development of chemically defined, animal origin-free (CD-AOF) media and supplements are unresolved issues that hamper the clinical applicability of these approaches. CD-AOF media and supplements ensure the quality and reproducibility of culture systems by lowering lot-to-lot variations and the risk of contamination with viruses or toxins. We previously generated liver organoids from iPSCs, namely iPSC-liver buds (iPSC-LBs), by mimicking the organogenic interactions among hepatocytes, endothelial cells (ECs), and mesenchymal cells (MCs) and recently reported the mass production of iPSC-LBs derived entirely from iPSCs (all iPSC-LBs), which should facilitate their large-scale production for the treatment of liver failure. However, in previous studies we used media originating from animals for differentiation except for the maintenance of undifferentiated iPSCs. Therefore, we developed a CD-AOF medium to generate all iPSC-LBs. We first developed a CD-AOF medium for hepatocytes, ECs, and stage-matched MCs, i.e., septum transversum mesenchyme (STM), in 2D cultures. We next generated all iPSC-LBs by incubating individual cell types in ultra-low attachment micro-dimple plates. The hepatic functions of all iPSC-LBs generated using the CD-AOF medium were equivalent to those of all iPSC-LBs generated using the conventional medium both in vitro and in vivo. Furthermore, we found that this CD-AOF medium could be used in several cell culture settings. Taken together, these results demonstrate the successful development of a CD-AOF medium suitable for all iPSC-LBs. The protocol developed in this study will facilitate the clinical applicability of all iPSC-LBs in the treatment of liver diseases.


2018 ◽  
Author(s):  
Fantuzzi Federica ◽  
Toivonen Sanna ◽  
Schiavo Andrea Alex ◽  
Pachera Nathalie ◽  
Rajaei Bahareh ◽  
...  

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