Development of a Method for Isolation of Mature Cardiomyocytes from Human Heart Biopsy Specimens.

To increase the yield of living cells and their survival, studies were carried out to optimize the method for isolating cardiomyocytes from biopsy specimens excised from the right atrial appendages. It was found that creatine, blebbistatin, and taurine are necessary components of the buffer solution during cardiomyocyte isolation, and that composition of the solutions is a more important factor than their oxygenation.

Conduction of excitation waves and reentry drift on cardiac tissue with simulated photocontrol-varied excitability.

The development of new approaches to suppressing cardiac arrhythmias requires a deep understanding of spiral wave dynamics. The study of spiral waves is possible in model systems, for example, in a monolayer of cardiomyocytes. A promising way to control cardiac excitability in vitro is the noninvasive photocontrol of cell excitability mediated by light-sensitive azobenzene derivatives, such as azobenzene trimethylammonium bromide (AzoTAB). The trans-isomer of AzoTAB suppresses spontaneous activity and excitation propagation speed, whereas the cis isomer has no detectable effect on the electrical properties of cardiomyocyte monolayers; cis isomerization occurs under the action of near ultraviolet (UV) light, and reverse isomerization occurs when exposed to blue light. Thus, AzoTAB makes it possible to create patterns of excitability in conductive tissue. Here, we investigate the effect of a simulated excitability gradient in cardiac cell culture on the behavior and termination of reentry waves. Experimental data indicate a displacement of the reentry wave, predominantly in the direction of lower excitability. However, both shifts in the direction of higher excitability and shift absence were also observed. To explain this effect, we reproduced these experiments in a computer model. Computer simulations showed that the explanation of the mechanism of observed drift to a lower excitability area requires not only a change in excitability coefficients (ion currents) but also a change in the diffusion coefficient; this may be the effect of the substance on intercellular connections. In addition, it was found that the drift direction depended on the observation time due to the meandering of the spiral wave. Thus, we experimentally proved the possibility of noninvasive photocontrol and termination of spiral waves with a mechanistic explanation in computer models.

Cyclophosphamide arrhythmogenicitytesting using human-induced pluripotent stem cell-derived cardiomyocytes.

Cyclophosphamide (CP) is an anticancer drug, an alkylating agent. Cardiotoxicity of CP is associated with one of its metabolites, acrolein, and clinical cardiotoxicity manifestations are described for cases of taking CP in high doses. Nevertheless, modern arrhythmogenicity prediction assays in vitro include evaluation of beat rhythm and rate as well as suppression of cardiac late markers after acute exposure to CP, but not its metabolites. The mechanism of CP side effects when taken at low doses (i.e., < 100 mg/kg), especially at the cellular level, remains unclear. In this study conduction properties and cytoskeleton structure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a healthy donor under CP were evaluated. Arrhythmogenicity testing including characterization of 3 values: conduction velocity, maximum capture rate (MCR) measurements and number of occasions of re-entry on a standard linear obstacle was conducted and revealed MCR decrease of 25% ± 7% under CP. Also, conductivity area reduced by 34 ± 15%. No effect of CP on voltage-gated ion channels was found. Conduction changes (MCR and conductivity area decrease) are caused by exposure time-dependent alpha-actinin disruption detected both in hiPSC-CMs and neonatal ventricular cardiomyocytes in vitro. Deviation from the external stimulus frequency and appearance of non-conductive areas in cardiac tissue under CP is potentially arrhythmogenic and could develop arrhythmic effects in vivo.

Formation of an electrical coupling between differentiating cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as an indispensable platform for the study of human cardiovascular disease is human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While the possibility of reproducing rare pathologies, patient-specific selection of drugs, and other issues concerning single cardiomyocytes have been well studied, little attention has been paid to the properties of the whole syncytium of CMs, in which both the functionality of individual cells and the distribution of electrophysiological connections between them are essential. The aim of this work is to directly study the ability of hiPSC-CMs to form a functional syncytium that can stably conduct an excitation wave. For that purpose, syncytium forming hiPSC-CMs were harvested and seeded (transferred) on a new substrate on different days of differentiation. The excitation conduction in a sample was characterized by the stability of the wavefront using optical mapping data. We found that the cells transferred before the 20th day of differentiation were able to organize a functional syncytium capable of further development and stable excitation conduction at high stimulation frequencies, while the cells transferred after 20 days did not form a homogeneous syncytium, and multiple instabilities of the propagating wavefront were observed with the possibility of reentry formation.

The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing.

Erythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5-100 µM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study, the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 µM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12 ± 9% at concentrations of 15-45 µM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28 ± 12% (p < 0.01). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a "window" of concentrations of the drug (15-30 µM) at which the probability of re-entry increased.

Effect of heptanol and ethanol on excitation wave propagation in a neonatal rat ventricular myocyte monolayer.

In this work, the action of heptanol and ethanol was investigated in a two-dimensional (2D) model of cardiac tissue: the neonatal rat ventricular myocyte monolayer. Heptanol is known in electrophysiology as a gap junction uncoupler but may also inhibit voltage-gated ionic channels. Ethanol is often associated with the occurrence of arrhythmias. These substances influence sodium, calcium, and potassium channels, but the complete mechanism of action of heptanol and ethanol remains unknown. The optical mapping method was used to measure conduction velocities (CVs) in concentrations of 0.05-1.8 mM heptanol and 17-1342 mM ethanol. Heptanol was shown to slow the excitation wave significantly, and a mechanism that involves a simultaneous action on cell coupling and activation threshold was suggested. Whole-cell patch-clamp experiments showed inhibition of sodium and calcium currents at a concentration of 0.5 mM heptanol. Computer modeling was used to estimate the relative contribution of the cell uncoupling and activation threshold increase caused by heptanol. Unlike heptanol, ethanol slightly influenced the CV at clinically relevant concentrations. Additionally, the critical concentrations for re-entry formation in ethanol were determined.

Synchronization of excitable cardiac cultures of different origin.

In the present work, we investigated the synchronization of electrical activity in cultured cardiac cells of different origin put in direct contact. In the first set of experiments synchronization was studied in the primary culture cells of neonatal rats taken at different developmental ages, and in the second - in the neonatal rat cardiomyocytes and HL-1 cells. The electrical excitation of cells was recorded using the calcium transient marker Fluor-4. In the confluent cell layers created with the aid of a specially devised mask, the excitation waves and their propagation between areas occupied by cells of different origin were observed. On the level of individual cells, their contact and synchronization was monitored with the aid of scanning fluorescence microscopy. It was found that populations of cultured cells of different origin are able to synchronize, suggesting the formation of electrical coupling between them. The results obtained may be considered as a proof of concept that implanted alien grafted cells are able to create electrical coupling with the host cardiac tissue.

Arrhythmogenic role of the border between two areas of cardiac cell alignment.

The goal of this study is to develop experimental and computational models of the excitation transition between areas of cardiac tissue with different anatomical anisotropy. Alignment of seeded neonatal rat cardiomyocytes was achieved with the aid of guiding polymer (PMGI) nanofibers, and two areas with orthogonal alignment were placed into a contact. It was found that the excitation wave crossing border between the areas with different alignment direction experiences substantial perturbation, up to the complete conduction block. In addition to the experimental study, this effect was analyzed computationally using generic FitzHugh-Nagumo reaction-diffusion model. It was shown that the non-monotonous changes of the excitation wave velocity on this boundary may be explained by the source/sink mismatch. Thus, the border may play pro-arrhythmogenic role.

Influence of patterned topographic features on the formation of cardiac cell clusters and their rhythmic activities.

In conventional primary cultures, cardiac cells prepared from a newborn rat undergo spontaneous formation of cell clusters after several days. These cell clusters may be non-homogeneously distributed on a flat surface and show irregular beating which can be recorded by calcium ion imaging. In order to improve the cell cluster homogeneity and the beating regularity, patterned topographic features were used to guide the cellular growth and the cell layer formation. On the substrate with an array of broadly spaced cross features made of photoresist, cells grew on the places that were not occupied by the crosses and thus formed a cell layer with interconnected cell clusters. Accordingly, spatially coordinated regular beating could be recorded over the whole patterned area. In contrast, when cultured on the substrate with broadly spaced but inter-connected cross features, the cardiac cell layer showed beatings which were neither coordinated in space nor regular in time. Finally, when cultured on the substrate with narrowly spaced features, the cell beating became spatially coordinated but still remained irregular. Our results suggest a way to improve the rhythmic property of cultured cardiac cell layers which might be useful for further investigations.

Curvature-Dependent Excitation Propagation in Cultured Cardiac Tissue.

The geometry of excitation wave front may play an important role on the propagation block and spiral wave formation. The wave front which is bent over the critical value due to interaction with the obstacles may partially cease to propagate and appearing wave breaks evolve into rotating waves or reentry. This scenario may explain how reentry spontaneously originates in a heart. We studied highly curved excitation wave fronts in the cardiac tissue culture and found that in the conditions of normal, non-inhibited excitability the curvature effects do not play essential role in the propagation. Neither narrow isthmuses nor sharp corners of the obstacles, being classical objects for production of extremely curved wave front, affect non-inhibited wave propagation. The curvature-related phenomena of the propagation block and wave detachment from the obstacle boundary were observed only after partial suppression of the sodium channels with Lidocaine. Computer simulations confirmed the experimental observations. The explanation of the observed phenomena refers to the fact that the heart tissue is made of finite size cells so that curvature radii smaller than the cardiomyocyte size loses sense, and in non-inhibited tissue the single cell is capable to transmit excitation to its neighbors.

Wave emission from heterogeneities opens a way to controlling chaos in the heart.

The effectiveness of chaos control in large systems increases with the number of control sites. We find that electric field induced wave emission from heterogeneities (WEH) in the heart gives a unique opportunity to have as many control sites as needed. The number of pacing sites grows with the amplitude of the electric field. We demonstrate that WEH has important advantages over methods used in clinics, and opens a new way to manipulate vortices in experiments, and potentially to radically improve the clinical methods of chaos control in the heart.

Light induced annihilation and shift of spiral waves.

The light-induced collapse of a pair of spiral waves was studied in a chemically active medium based on the photosensitive Ru(bpy)(3)-catalyzed Belousov-Zhabotinsky reaction. Spiral waves annihilate only if the light intensity is increased in proper phase relative to the spiral waves' rotation. Otherwise, the distance between spiral wave cores increases and the pair survives. Computer simulations reveal the mechanism which forces the spiral waves to collide and annihilate. It is based on the shift of a single spiral wave upon an instantaneous decrease of excitability of the medium. (c) 1996 American Institute of Physics.

High-frequency instability of wave fronts.

Experimental evidence is presented that a lateral instability of a wave front, as described earlier in a chemically active medium with the Belousov-Zhabotinsky reaction with decreased excitability, can also occur in a medium with any degree of excitability provided that a high-frequency wave train travels through the medium. The interaction of chemical waves with the boundary of the medium can result in the appearance of wave breaks and spiral waves.

Rotating spiral waves created by geometry.

The Belousov-Zhabotinsky reagent and numerical simulations were used to show that under high-frequency stimuli, rotating spiral waves can be initiated in a homogeneous excitable medium in the vicinity of domain boundaries or inexcitable barriers with sharp corners.

Wave mechanisms of pattern formation in microbial populations.

The occurrence of spatially ordered structures plays an important role in biology (examples: morphogenesis, ecosystems, dynamics of populations, etc.). Turing proposed a reaction-diffusion process that is the basis for most theoretical studies of stationary biological pattern formation. Now, when Turing structures are obtained in experiments (40 years after Turing's publication), it is interesting to discover whether Turing structures are the only mechanism used by nature in biological pattern formation. In microbial growth, we have found experimental evidence of an alternative to the Turing model that is based on waves displayed in excitable media. In studies of Escherichia coli populations, we observed that interacting taxis waves create motionless patterns. Taxis waves consuming two different substrates (serine and aspartic acid) were involved. Taxis waves consuming serine stop when they collide. However, those supported by consumption of aspartic were initiated at the collision line. Colliding and annihilating in turn, the waves give rise to stationary pattern formation, and wave theory provides an alternative to the classical Turing mechanism.