Electrophysiological evaluation of pentamidine and 17-AAG in human stem cell-derived cardiomyocytes for safety assessment.

Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs' actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.

On-chip spatiotemporal electrophysiological analysis of human stem cell derived cardiomyocytes enables quantitative assessment of proarrhythmia in drug development.

We examined a simultaneous combined spatiotemporal field potential duration (FPD) and cell-to-cell conduction time (CT) in lined-up shaped human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using an on-chip multielectrode array (MEA) system to evaluate two origins of lethal arrhythmia, repolarization and depolarization. The repolarization index, FPD, was prolonged by E-4031 and astemizole, and shortened by verapamil, flecainide and terfenadine at 10 times higher than therapeutic plasma concentrations of each drug, but it did not change after lidocaine treatment up to 100 µM. CT was increased by astemizol, flecainide, terfenadine, and lidocaine at equivalent concentrations of Nav1.5 IC50, suggesting that CT may be an index of cardiac depolarization because the increase in CT (i.e., decrease in cell-to-cell conduction speed) was relevant to Nav1.5 inhibition. Fluctuations (short-term variability; STV) of FPD and CT, STVFPD and STVCT also discriminated between torsadogenic and non-torsadogenic compounds with significant increases in their fluctuation values, enabling precise prediction of arrhythmogenic risk as potential new indices.

Integrate and fire model with refractory period for synchronization of two cardiomyocytes.

We investigate an integrate and fire model for two cardiomyocytes interacting with each other. A feature of the model is to incorporate the refractory periods of the cardiomyocytes as well as the influence of firing of adjacent cells. The present model predicts that, if refractory periods of the two cells are nearly equal, the beating rhythms of the two cells always synchronize and their beating rate is tuned to the faster rate between the two cells. On the other hand, if their refractory periods significantly differ, they exhibit various kinds of harmonious beating rhythms. These results successfully explain the well known characteristics of synchronized beating of cultured cardiomyocytes. We also discuss effects of a delay time of cell-to-cell interaction, that gives further complicated phase diagrams for the beating rhythms.

A distribution analysis of action potential parameters obtained from patch-clamped human stem cell-derived cardiomyocytes.

We investigated electrophysiological properties of human induced-pluripotent-stem-cell-derived and embryonic-stem-cell-derived cardiomyocytes, and analyzed action potential parameters by plotting their frequency distributions. In the both cell lines, the distribution analysis revealed that histograms of maximum upstroke velocity showed two subpopulations with similar intersection values. Sub-populations with faster maximum upstroke velocity showed significant prolongation of action potential durations by application of E-4031, whereas others did not, which may be partly due to shallower maximum diastolic potentials. We described electrophysiological and pharmacological properties of stem-cell-derived cardiomyocytes in the respective sub-populations, which provides a way to characterize diverse electrical properties of stem-cell-derived cardiomyocytes systematically.

Effects of lipid composition and solution conditions on the mechanical properties of membrane vesicles.

The mechanical properties of cell-sized giant unilamellar liposomes were studied by manipulating polystyrene beads encapsulated within the liposomes using double-beam laser tweezers. Mechanical forces were applied to the liposomes from within by moving the beads away from each other, which caused the liposomes to elongate. Subsequently, a tubular membrane projection was generated in the tip at either end of the liposome, or the bead moved out from the laser trap. The force required for liposome transformation reached maximum strength just before formation of the projection or the moving out of the bead. By employing this manipulation system, we investigated the effects of membrane lipid compositions and environment solutions on the mechanical properties. With increasing content of acidic phospholipids, such as phosphatidylglycerol or phosphatidic acid, a larger strength of force was required for the liposome transformation. Liposomes prepared with a synthetic dimyristoylphosphatidylcholine, which has uniform hydrocarbon chains, were transformed easily compared with liposomes prepared using natural phosphatidylcholine. Surprisingly, bovine serum albumin or fetuin (soluble proteins that do not bind to membranes) decreased liposomal membrane rigidity, whereas the same concentration of sucrose showed no particular effect. These results show that the mechanical properties of liposomes depend on their lipid composition and environment.

On-chip in vitro cell-network pre-clinical cardiac toxicity using spatiotemporal human cardiomyocyte measurement on a chip.

To overcome the limitations and misjudgments of conventional prediction of arrhythmic cardiotoxicity, we have developed an on-chip in vitro predictive cardiotoxicity assay using cardiomyocytes derived from human stem cells employing a constructive spatiotemporal two step measurement of fluctuation (short-term variability; STV) of cell's repolarization and cell-to-cell conduction time, representing two origins of lethal arrhythmia. Temporal STV of field potential duration (FPD) showed a potential to predict the risks of lethal arrhythmia originated from repolarization dispersion for false negative compounds, which was not correctly predicted by conventional measurements using animal cells, even for non-QT prolonging clinical positive compounds. Spatial STV of conduction time delay also unveiled the proarrhythmic risk of asynchronous propagation in cell networks, whose risk cannot be correctly predicted by single-cell-based measurements, indicating the importance of the spatiotemporal fluctuation viewpoint of in vitro cell networks for precise prediction of lethal arrhythmia reaching clinical assessment such as thorough QT assay.

Microtechnologies to fuel neurobiological research with nanometer precision.

The interface between engineering and molecular life sciences has been fertile ground for advancing our understanding of complex biological systems. Engineered microstructures offer a diverse toolbox for cellular and molecular biologists to direct the placement of cells and small organisms, and to recreate biological functions in vitro: cells can be positioned and connected in a designed fashion, and connectivity and community effects of cells studied. Because of the highly polar morphology and finely compartmentalized functions of neurons, microfabricated cell culture systems and related on-chip technologies have become an important enabling platform for studying development, function and degeneration of the nervous system at the molecular and cellular level. Here we review some of the compartmentalization techniques developed so far to highlight how high-precision control of neuronal connectivity allows new approaches for studying axonal and synaptic biology.

Multiple membrane interactions and versatile vesicle deformations elicited by melittin.

Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the "increasing membrane area", "phased shrinkage", or "solubilization" of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, ß-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions.

Exploring the implicit interlayer regulatory mechanism between cells and tissue: stochastic mathematical analyses of the spontaneous ordering in beating synchronization.

The present study focused on beating synchronization, and tried to elucidate the interlayer regulatory mechanisms between the cells and clump in beating synchronization with using the stochastic simulations which realize the beating synchronizations in beating cells with low cell-cell conductance. Firstly, the fluctuation in interbeat intervals (IBIs) of beating cells encouraged the process of beating synchronization, which was identified as the stochastic resonance. Secondly, fluctuation in the synchronized IBIs of a clump decreased as the number of beating cells increased. The decrease in IBI fluctuation due to clump formation implied both a decline of the electrophysiological plasticity of each beating cell and an enhancement of the electrophysiological stability of the clump. These findings were identified as the community effects. Because IBI fluctuation and the community effect facilitated the beating stability of the cell and clump, these factors contributed to the spontaneous ordering in beating synchronization. Thirdly, the cellular layouts in clump affected the synchronized beating rhythms. The synchronized beating rhythm in clump was implicitly regulated by a complicated synergistic effect among IBI fluctuation of each beating cell, the community effect and the cellular layout. This finding was indispensable for leading an elucidation of mechanism of emergence. The stochastic simulations showed the necessity of considering the synergistic effect, to elucidate the interlayer regulatory mechanisms in biological system.

A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES) cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

On-chip constructive cell-network study (II): on-chip quasi-in vivo cardiac toxicity assay for ventricular tachycardia/fibrillation measurement using ring-shaped closed circuit microelectrode with lined-up cardiomyocyte cell network.

BACKGROUNDS: Conventional in vitro approach using human ether-a-go-go related gene (hERG) assay has been considered worldwide as the first screening assay for cardiac repolarization safety. However, it does not always oredict the potential QT prolongation risk or pro-arrhythmic risk correctly. For adaptable preclinical strategiesto evaluate global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal arrhythmia (ventricular tachyarrhythmia) measurement using ring-shaped closed circuit microelectrode chip has been developed. RESULTS: The ventricular electrocardiogram (ECG)-like field potential data, which includes both the repolarization and the conductance abnormality, was acquired from the self-convolutied extracellular field potentials (FPs) of a lined-up cardiomyocyte network on a circle-shaped microelectrode in an agarose microchamber. When Astemisol applied to the closed-loop cardiomyocyte network, self-convoluted FP profile of normal beating changed into an early afterdepolarization (EAD) like waveform, and then showed ventricular tachyarrhythmias and ventricular fibrilations (VT/Vf). QT-prolongation-like self-convoluted FP duration prolongation and its fluctuation increase was also observed according to the increase of Astemizole concentration. CONCLUSIONS: The results indicate that the convoluted FPs of the quasi-in vivo cell network assay includes both of the repolarization data and the conductance abnormality of cardiomyocyte networks has the strong potential to prediction lethal arrhythmia.

On-chip constructive cell-network study (I): contribution of cardiac fibroblasts to cardiomyocyte beating synchronization and community effect.

BACKGROUNDS: To clarify the role of cardiac fibroblasts in beating synchronization, we have made simple lined-up cardiomyocyte-fibroblast network model in an on-chip single-cell-based cultivation system. RESULTS: The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts showed (1) propagation velocity of electrophysiological signals decreased a magnitude depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2) fluctuation of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and (3) the synchronized cardiomyocytes connected by fibroblasts sometimes loses their synchronized condition and recovered to synchronized condition, in which the length of asynchronized period was shorter less than 30 beats and was independent to their cultivation time, whereas the length of synchronized period increased according to cultivation time. CONCLUSIONS: The results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly enhance and contribute to beating interval stability and synchronization. This might also mean that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte 'community effect', which enhances synchronization and stability of their beating rhythms.

In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes.

The lethal ventricular arrhythmia Torsade de pointes (TdP) is the most common reason for the withdrawal or restricted use of many cardiovascular and non-cardiovascular drugs. The lack of an in vitro model to detect pro-arrhythmic effects on human heart cells hinders the development of new drugs. We hypothesized that recently established human induced pluripotent stem (hiPS) cells could be used in an in vitro drug screening model. In this study, hiPS cells were driven to differentiate into functional cardiomyocytes, which expressed cardiac markers including Nkx2.5, GATA4, and atrial natriuretic peptide. The hiPS-derived cardiomyocytes (hiPS-CMs) were analyzed using a multi electrode assay. The application of ion channel inhibitors resulted in dose-dependent changes to the field potential waveform, and these changes were identical to those induced in the native cardiomyocytes. This study shows that hiPS-CMs represent a promising in vitro model for cardiac electrophysiologic studies and drug screening.

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force.

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.

Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion.

To study the mechanisms involved in membrane fusion, we visualized the fusion process of giant liposomes in real time by optical dark-field microscopy. To induce membrane fusion, we used (i) influenza hemagglutinin peptide (HA), a 20-aa peptide derived from the N-terminal fusion peptide region of the HA2 subunit, and (ii) two synthetic analogue peptides of HA, a negatively (E5) and positively (K5) charged analogue. We were able to visualize membrane fusion caused by E5 or by K5 alone, as well as by the mixture of these two peptides. The HA peptide however, did not induce membrane fusion, even at an acidic pH, which has been described as the optimal condition for the fusion of large unilamellar vesicles. Surprisingly, before membrane fusion, the shrinkage of liposomes was always observed. Our results suggest that a perturbation of lipid bilayers, which probably resulted from alterations in the bending folds of membranes, is a critical factor in fusion efficiency.

Liposomes possess drastic capabilities for topological transformation.

Morphological and topological changes of biological membranes play essential roles in cellular activities. It has been thought that these transformations are made possible through interactions with proteins. However, direct observation of giant liposomes by optical dark-field microscopy reveals that the lipid bilayer itself possesses the ability to undergo topological transformation.