Tetsuro Wakatsuki

Cardiomyocytes continuously experience mechanical stimuli that regulate their contractile behavior and contribute to overall heart function. Despite the importance of mechanotransduction in cardiac physiology, the mechanisms by which cardiomyocytes integrate external mechanical cues, such as stretch and environmental stiffness, remain poorly understood. In this study, we present a combined theoretical and experimental framework to investigate how strain-induced cytoskeletal stiffening modulates cardiomyocyte contractility and force generation. Our study elucidates that regulating the mechanical tension cardiomyocytes experience in tissue—whether by modulating environmental stiffness, external stretching, or cardiac fibroblast activation—can effectively tune their contractility, with cytoskeletal strain stiffening playing a central role in this mechanotransductive response.

Among various cardiac safety concerns, proarrhythmia risks, including QT prolongation leading to Torsade de Pointes, is one of major cause for drugs being withdrawn (~45% 1975–2007). Preclinical study requires the evaluation of proarrhythmia using in silico, in vitro, and/or animal models. Considering that the primary consumers of prescription drugs are elderly patients, applications of “aging-in-a-dish” models would be appropriate for screening proarrhythmia risks. However, acquiring such models, including cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs), presents extensive challenges. We proposed the hypothesis that CMs differentiated from iPSCs derived from Hutchinson–Gilford progeria syndrome (HGPS, progeria) patients, an ultra-rare premature aging syndrome, can mimic the phenotypes of aging CMs. Our objective, therefore, was to examine this hypothesis by analyzing the response of 11 reference compounds utilized by the Food and Drug Administration (FDA)’s Comprehensive in vitro Proarrhythmia Assay (CiPA) using progeria and control CMs. As a sensitive surrogate marker of modulating cardiac excitation–contraction coupling, we evaluated drug-induced changes in calcium transient (CaT). We observed that the 80% CaT peak duration in the progeria CMs (0.98 ± 0.04 s) was significantly longer than that of control CMs (0.70 ± 0.05 s). Furthermore, when the progeria CMs were subjected to four doses of 11 compounds from low-, intermediate-, and high-risk categories, they demonstrated greater arrhythmia susceptibility than control cells, as shown through six-parameter CaT profile analyses. We also employed the regression analysis established by CiPA to classify the 11 reference compounds and compared proarrhythmia susceptibilities between the progeria and control CMs. This analysis revealed a greater proarrhythmia susceptibility in the progeria CMs compared to the control CMs. Interestingly, in both CMs, the compounds categorized as low risk did not exceed the safety risk threshold of 0.8. In conclusion, our study demonstrates increased proarrhythmia sensitivity in progeria CMs when tested with reference compounds. Future studies are needed to analyze underlying mechanisms and further validate our findings using a larger array of reference compounds.

The one-size-fits-all approach has been the mainstream in medicine, and the well-defined standards support the development of safe and effective therapies for many years. Advancing technologies, however, enabled precision medicine to treat a targeted patient population (e.g., HER2+ cancer). In safety pharmacology, computational population modeling has been successfully applied in virtual clinical trials to predict drug-induced proarrhythmia risks against a wide range of pseudo cohorts. In the meantime, population modeling in safety pharmacology experiments has been challenging. Here, we used five commercially available human iPSC-derived cardiomyocytes growing in 384-well plates and analyzed the effects of ten potential proarrhythmic compounds with four concentrations on their calcium transients (CaTs). All the cell lines exhibited an expected elongation or shortening of calcium transient duration with various degrees. Depending on compounds inhibiting several ion channels, such as hERG, peak and late sodium and L-type calcium or IKs channels, some of the cell lines exhibited irregular, discontinuous beating that was not predicted by computational simulations. To analyze the shapes of CaTs and irregularities of beat patterns comprehensively, we defined six parameters to characterize compound-induced CaT waveform changes, successfully visualizing the similarities and differences in compound-induced proarrhythmic sensitivities of different cell lines. We applied Bayesian statistics to predict sample populations based on experimental data to overcome the limited number of experimental replicates in high-throughput assays. This process facilitated the principal component analysis to classify compound-induced sensitivities of cell lines objectively. Finally, the association of sensitivities in compound-induced changes between phenotypic parameters and ion channel inhibitions measured using patch clamp recording was analyzed. Successful ranking of compound-induced sensitivity of cell lines was validated by visual inspection of raw data. Author Summary Cardiac safety is one of the most stringent regulatory risk monitoring during drug development. Regulatory agencies, including the Food and Drug Administration (FDA), require drug developers to conduct thorough preclinical and clinical studies to evaluate drug-induced proarrhythmia risks. The CiPA (Comprehensive in vitro Proarrhythmia Assay) initiative led by the FDA validated the applications of human cardiomyocytes derived from induced pluripotent stem cells in proarrhythmia risk assessments. The assay can be cost effective, and use of high-throughput approaches enables scale-up analysis. However, limited diversity in cell lines to recapitulate heterogeneity of human patients are still lacking in the cardiac safety analysis. We applied various computational tools to maximize capacity for analyzing diverse response of commercially available five cell lines against reference chemical compounds. Limited number of experimental data made it difficult to predict similarities and differences in drug responses among different cell lines. By applying Bayesian approach, our analysis could intuitively grasp the probabilities of expecting different responses from different cell lines.

Heart failure is the leading cause of death in the western world and as such, there is a great need for new therapies. Heart failure has a variable presentation in patients and a complex etiology; however, it is fundamentally a condition that affects the mechanics of cardiac contraction, preventing the heart from generating sufficient cardiac output under normal operating pressures. One of the major issues hindering the development of new therapies has been difficulties in developing appropriate in vitro model systems of human heart failure that recapitulate the essential changes in cardiac mechanics seen in the disease. Recent advances in stem cell technologies, genetic engineering, and tissue engineering have the potential to revolutionize our ability to model and study heart failure in vitro. Here, we review how these technologies are being applied to develop personalized models of heart failure and discover novel therapeutics.

A mathematical model describing the electrophysiology and ion handling of cardiomyocytes is a complement to experimental analysis predicting drug-induced proarrhythmic potential in humans as proposed by Comprehensive in vitro Proarrhythmia Assay (CiPA). While CiPA endorses the use of the O’Hara Rudy (ORd) model, which was developed to simulate electrophysiology of human adult ventricular cardiomyocytes (hAVCMs), to predict drug-induced proarrhythmias; the human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) was proposed for experimental verifications. The hiPSC-CMs, especially cultured in 2D culture dishes, are inherently different from hAVCMs exhibiting different ion channel density and an immature sarcoplasmic reticulum function. To reconcile this mismatch, we have developed a mathematical electrophysiology model of an hiPSC-CM by incorporating differences in gene expressions of ion channels, pumps and receptors in hiPSC-CMs against those found in hAVCMs. This model can be used to model any hiPSC-CM cell line where expression data has been obtained and replaces the background currents for K ⁺ and Na ⁺ in the ORd model with the known ultra-rapid K ⁺ channel and hyperpolarization activated K ⁺ /Na ⁺ channel currents. With this new model, three batches each from two different hiPSC-CM cell lines are compared to experimental data of action potential duration. This mathematical model recapitulates a ventricular-like action potential morphology with a Phase 2 plateau lasting a few hundred milliseconds. However, the resting membrane potential is not as depolarized (−65 to −70 mV) as that of hAVCM (-80 mV). The elevated resting membrane potential matches experimental data previously and is thought to keep rapid sodium channels from triggering membrane depolarization instead being replaced by an overexpression of L-Type Ca ²⁺ channel current. This model is a key step in identifying the variability between different hiPSC-CM lines and even batches of the same line, opening the door to realizing analysis of patient specific preparations in the future.

Electrophysiology of excitable cells, including muscle cells and neurons, has been measured by making direct contact with a single cell using a micropipette electrode. To increase the assay throughput, optical devices such as microscopes and microplate readers have been used to analyze electrophysiology of multiple cells. We have established a high-throughput (HTP) analysis of action potentials (APs) in highly enriched motor neurons and cardiomyocytes (CMs) that are differentiated from human induced pluripotent stem cells (iPSCs). A multichannel electric field stimulation (EFS) device enabled the ability to electrically stimulate cells and measure dynamic changes in APs of excitable cells ultra-rapidly (>100 data points per second) by imaging entire 96-well plates. We found that the activities of both neurons and CMs and their response to EFS and chemicals are readily discerned by our fluorescence imaging-based HTP phenotyping assay. The latest generation of calcium (Ca ²⁺ ) indicator dyes, FLIPR Calcium 6 and Cal-520, with the HTP device enables physiological analysis of human iPSC-derived samples highlighting its potential application for understanding disease mechanisms and discovering new therapeutic treatments.

Calcium (Ca2+) plays a central role in regulating many biological processes in the cell from muscle contraction to neurotransmitter release. The need for reliable fluorescent calcium indicator dyes is of vast importance for studying many aspects of cell biology as well as screening compounds using phenotypic high throughput assays. We have assessed two of the latest generation of calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM for studying calcium transients (CaTs) in induced pluripotent stem cell (iPSC) -derived human cardiomyocytes. FLIPR Calcium 6 and Cal-520 dyes both displayed robust CaTs with a high signal-to-noise ratio (SNR) and were non-toxic to the cells. The analysis showed that CaT amplitudes were stable between measurements, but CaT duration was more variable and tended to increase between reads. Two methods were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference. The unstandardized difference was better for assessing CaT amplitude, whereas standardized difference was equal to or better for assessing CaT duration. In summary, FLIPR Calcium 6 and Cal-520 are suitable dyes for drug-screening using iPSC-derived human cardiomyocytes.

Pluripotent Stem Cells were originally derived and cultured using a feeder layer of cells. Movements have been undertaken to transition from this method to one more defined, high-throughput, and without xenogenic factors. Tremendous research has been done in this area and many products have been developed, however, based on our analysis of recent publications in stem cell related journals many in academia are still using older methods like a feeder layer. In this short communication, we discuss the feasibility of transitioning to defined, xeno-free methods, how a standardized method could improve the field and industry, and that a study bringing together multiple institutions comparing culture methods could be done to evaluate the efficacy of these new methods.