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Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell-derived cardiomyocytes.

ABSTRACT: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell-cell and cell-substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers (1) attached to glass or plastic (Young's modulus (E) >1 GPa), (2) detached (substrate-free) and (3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress-activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions. KEY POINTS: Spatiotemporal contractility analysis of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical. These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only 'twitch'-like transients are observed. HiPSC-CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response. There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress-activated NPPA or NPPB signalling pathways. A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell-substrate adhesion underly multiphasic contractile behaviour of hiPSC-CMs.

SUBMITTER: Huethorst E

PROVIDER: S-EPMC9299844 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsHuman-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 resultsAction 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.ConclusionTaking 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.

Project description:Stem cell therapy has the potential to regenerate cardiac function after myocardial infarction. In this study, we sought to examine if fibrin microthread technology could be leveraged to develop a contractile fiber from human pluripotent stem cell derived cardiomyocytes (hPS-CM). hPS-CM seeded onto fibrin microthreads were able to adhere to the microthread and began to contract seven days after initial seeding. A digital speckle tracking algorithm was applied to high speed video data (>60 fps) to determine contraction behaviour including beat frequency, average and maximum contractile strain, and the principal angle of contraction of hPS-CM contracting on the microthreads over 21 days. At day 7, cells seeded on tissue culture plastic beat at 0.83 ± 0.25 beats/sec with an average contractile strain of 4.23±0.23%, which was significantly different from a beat frequency of 1.11 ± 0.45 beats/sec and an average contractile strain of 3.08±0.19% at day 21 (n = 18, p < 0.05). hPS-CM seeded on microthreads beat at 0.84 ± 0.15 beats/sec with an average contractile strain of 3.56±0.22%, which significantly increased to 1.03 ± 0.19 beats/sec and 4.47±0.29%, respectively, at 21 days (n = 18, p < 0.05). At day 7, 27% of the cells had a principle angle of contraction within 20 degrees of the microthread, whereas at day 21, 65% of hPS-CM were contracting within 20 degrees of the microthread (n = 17). Utilizing high speed calcium transient data (>300 fps) of Fluo-4AM loaded hPS-CM seeded microthreads, conduction velocities significantly increased from 3.69 ± 1.76 cm/s at day 7 to 24.26 ± 8.42 cm/s at day 21 (n = 5-6, p < 0.05). hPS-CM seeded microthreads exhibited positive expression for connexin 43, a gap junction protein, between cells. These data suggest that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction. In addition, extended culture allows cells to contract in the direction of the thread, suggesting alignment of the cells in the microthread direction.

Project description:BackgroundDespite the accumulating genetic and molecular investigations into hypertrophic cardiomyopathy (HCM), it remains unclear how this condition develops and worsens pathologically and clinically in terms of the genetic-environmental interactions. Establishing a human disease model for HCM would help to elucidate these disease mechanisms; however, cardiomyocytes from patients are not easily obtained for basic research. Patient-specific induced pluripotent stem cells (iPSCs) potentially hold much promise for deciphering the pathogenesis of HCM. The purpose of this study is to elucidate the interactions between genetic backgrounds and environmental factors involved in the disease progression of HCM.Methods and resultsWe generated iPSCs from 3 patients with HCM and 3 healthy control subjects, and cardiomyocytes were differentiated. The HCM pathological phenotypes were characterized based on morphological properties and high-speed video imaging. The differences between control and HCM iPSC-derived cardiomyocytes were mild under baseline conditions in pathological features. To identify candidate disease-promoting environmental factors, the cardiomyocytes were stimulated by several cardiomyocyte hypertrophy-promoting factors. Interestingly, endothelin-1 strongly induced pathological phenotypes such as cardiomyocyte hypertrophy and intracellular myofibrillar disarray in the HCM iPSC-derived cardiomyocytes. We then reproduced these phenotypes in neonatal cardiomyocytes from the heterozygous Mybpc3-targeted knock in mice. High-speed video imaging with motion vector prediction depicted physiological contractile dynamics in the iPSC-derived cardiomyocytes, which revealed that self-beating HCM iPSC-derived single cardiomyocytes stimulated by endothelin-1 showed variable contractile directions.ConclusionsInteractions between the patient's genetic backgrounds and the environmental factor endothelin-1 promote the HCM pathological phenotype and contractile variability in the HCM iPSC-derived cardiomyocytes.

Project description:Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal relationship between electrical excitation and contractile motion, we simultaneously observed the field potential and motion vector of hiPSC-CMs sheets. Under spontaneous contraction, although an electrical excitation originating from a region propagated unidirectionally over the cell sheet, contraction wave started from multiple sites, and relaxation wave was initiated from a geometric center of hiPSC-CMs sheet. During electrical pacing, contraction and relaxation waves were propagated from the stimulated site. Interestingly, the maximum contraction speed was more increased when the hiPSC-CMs sheet was stimulated at an area relaxation initiated under spontaneous condition. Furthermore, motion vector analysis demonstrated that "positive contraction velocity-frequency relationship" in contraction and "frequency-dependent enhancement of relaxation" were produced in the cell sheet by optimizing the direction and order of the contractile motion with pacing at the relaxation-initiating area. A close analysis of motion vectors along with field potential recording demonstrated that relaxation process consists of fast and slow phases, and suggest that intracellular Ca2+ dynamics may be closely related to functions of Ca2+-ATPase pump and Na+-Ca2+ exchangers. Namely, the slow relaxation phase occurred after the second peak of field potential, suggesting that the slow phase may be associated with extrusion of Ca2+ by Na+-Ca2+ exchangers during repolarization. Increase of oxygen concentration from 20 to 95% as well as β-adrenergic stimulation with isoproterenol accelerated the fast relaxation, suggesting that it could depend on Ca2+ uptake via Ca2+-ATPase during the depolarization phase. The ratio of maximum contraction speed to field potential duration was increased by the β-adrenergic stimulation, indicating the elevated contraction efficiency per Ca2+-influx. Thus, these findings revealed potential ability of conventional monolayers of hiPSC-CMs, which will help apply them to translational study filling the gap between physiological as well as pharmacological studies and clinical practice.

Project description:Background and purposeIn the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity.Experimental approachVideo microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment.Key resultsDifferent cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression.Conclusion and implicationsWe have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery.Linked articlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

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Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell-derived cardiomyocytes.

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