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Botulinum toxin A is a well-known neurotransmitter inhibitor with a wide range of applications in modern medicine. Recently, botulinum toxin A preparations have been used in clinical trials to suppress cardiac arrhythmias, especially in the postoperative period. Its antiarrhythmic action is associated with inhibition of the nervous system of the heart, but its direct effect on heart tissue remains unclear. Accordingly, we investigate the effect of botulinum toxin A on isolated cardiac cells and on layers of cardiac cells capable of conducting excitation. Cardiomyocytes of neonatal rat pups and human cardiomyocytes obtained through cell reprogramming were used. A patch-clamp study showed that botulinum toxin A inhibited fast sodium currents and L-type calcium currents in a dose-dependent manner, with no apparent effect on potassium currents. Optical mapping showed that in the presence of botulinum toxin A, the propagation of the excitation wave in the layer of cardiac cells slows down sharply, conduction at high concentrations becomes chaotic, but reentry waves do not form. The combination of botulinum toxin A with a preparation of chitosan showed a stronger inhibitory effect by an order of magnitude. Further, the inhibitory effect of botulinum toxin A is not permanent and disappears after 12 days of cell culture in a botulinum toxin A-free medium. The main conclusion of the work is that the antiarrhythmic effect of botulinum toxin A found in clinical studies is associated not only with depression of the nervous system but also with a direct effect on heart tissue. Moreover, the combination of botulinum toxin A and chitosan reduces the effective dose of botulinum toxin A.

Myocardial remodeling is an inevitable risk factor for cardiac arrhythmias and can potentially be corrected with cell therapy. Although the generation of cardiac cells ex vivo is possible, specific approaches to cell replacement therapy remain unclear. On the one hand, adhesive myocyte cells must be viable and conjugated with the electromechanical syncytium of the recipient tissue, which is unattainable without an external scaffold substrate. On the other hand, the outer scaffold may hinder cell delivery, for example, making intramyocardial injection difficult. To resolve this contradiction, we developed molecular vehicles that combine a wrapped (rather than outer) polymer scaffold that is enveloped by the cell and provides excitability restoration (lost when cells were harvested) before engraftment. It also provides a coating with human fibronectin, which initiates the process of graft adhesion into the recipient tissue and can carry fluorescent markers for the external control of the non-invasive cell position. In this work, we used a type of scaffold that allowed us to use the advantages of a scaffold-free cell suspension for cell delivery. Fragmented nanofibers (0.85 µm ± 0.18 µm in diameter) with fluorescent labels were used, with solitary cells seeded on them. Cell implantation experiments were performed in vivo. The proposed molecular vehicles made it possible to establish rapid (30 min) electromechanical contact between excitable grafts and the recipient heart. Excitable grafts were visualized with optical mapping on a rat heart with Langendorff perfusion at a 0.72 ± 0.32 Hz heart rate. Thus, the pre-restored grafts’ excitability (with the help of a wrapped polymer scaffold) allowed rapid electromechanical coupling with the recipient tissue. This information could provide a basis for the reduction of engraftment arrhythmias in the first days after cell therapy.

In the last decade,invitroexperiments have shown that mechanical properties of the bases could markedly influence the efficacy of differentiation of the induced pluripotent and embryonic stem cells and their development into the mature phenotype. By changing of mechanical, elastic and structural characteristics of the base, it is possible to increase the percentage of stem cells that differentiate to cardiomyocytes. The study was aimed at evaluation of the effects induced by changing physical characteristics of the base on the formation of phenotypic characteristics of cardiac cells. This included the comparison of structural properties of the cultured layer of heart tissue obtained by changing of elasticity and structure of polymeric bases. The results showed significant differences in calcium activity and structural characteristics of cardiomyocytes depending on the base properties, as well as significant variation in the excitation conduction. As long as conduction abnormalities in the heart tissues can often lead to occurrence of life-threatening cardiac arrhythmias, the results can be used to determine patient groups at increased risk of death from heart failure.

Botulinum toxin A is well known as a neurotransmitter inhibitor with a wide range of applications in modern medicine. In recent years, botulinum toxin preparations have begun to be used in clinical trials to suppress cardiac arrhythmias, especially in the postoperative period. Its antiarrhythmic action was associated with inhibition of the nervous system of the heart, while the question of a direct effect on the heart tissue remained unclear. In our work, we investigated the effect of botulinum toxin both on isolated cardiac cells and on layers of cardiac cells capable of conducting excitation. As an object of research, cardiomyocytes of neonatal rat pups and human cardiomyocytes obtained by the method of cell reprogramming were used. A patch-clamp study showed that botulinum toxin inhibited fast sodium current and L-type calcium current in a dose-dependent manner, with no apparent effect on potassium currents. It was shown by optical mapping that in the presence of botulinum toxin, the propagation of the excitation wave in the layer of cardiac cells slows down sharply, conduction at high concentrations becomes chaotic, but reentry waves do not form. It was found that the combination of botulinum toxin with a preparation of chitosan has an order of magnitude stronger inhibitory effect. It has been shown that the inhibitory effect of botulinum toxin is not permanent and disappears after 12 days of cell culture in a botulinum toxin-free medium.

Muscular thin films (MTFs), have already found a variety of applications in cardiac tissue engineering and in building of lab-on-a-chip systems. Here we present a novel approach to label-free mapping of excitation waves in the cardiomyocyte cell cultures with the use of MTFs. Neonatal rat ventricular cardiomyocytes were cultured on polydimethylsiloxane (PDMS) thin films and observed by means of off-axis illumination. Inflexions of the membrane created by the contraction of cardiomyocytes led to formation of patterns of bright and dark areas on the surface of the membrane. These patterns were recorded and analyzed for the monitoring of the contraction propagation. The method was compared with a standard optical mapping technique based on the use of a Ca2+-sensitive fluorescent dye. A good consistency of the results obtained by these two methods was demonstrated. The proposed method is non-toxic and might be of particular interest for the purpose of continuous monitoring in test systems based on human induced pluripotent stem cells.

The methods of cardiac conduction system recovery with the use of reprogrammed cells and biomaterials, which could provide a stable physiological heart rate throughout the lifetime, are currently studied for possible applications in the heart rhythm disorder treatment. The main issue of intramyocardial transplantation is the transplanted cells' survival and electrophysiological connections formation with the recipient cardiomyocytes. In this paper, in search for working approaches for creating an optical biological pacemaker, we studied the effectiveness of various methods of photosensitive cardiac ChR2-HL-1 line cells integration into neonatal rat cardiac monolayers. For the study, two approaches of co-cultivation were selected: isolated cells and clusters integration of the Ch2-HL-1 line in various concentrations into monolayers. The effectiveness of the obtained model pacemaker was evaluated by the following means: registration of excitation waves initiated by optical stimulation, which affected only cells of the Ch2-HL-1 line, was carried out by optical mapping; immunocytochemical analysis methods were used to characterize the morphology of the obtained co-cultures and to assess the degree of the integration of the embedded structures into the monolayer. In the course of the research it was shown that the most effective method of integration of cells in the primary culture is the cluster method: 100 % of samples with clusters, integrated after 6 hours of monolayer cultivation, showed stable generation of excitation waves at physiologically significant external stimulation frequencies, compared with 88 % for specimens with integrated isolated cells at frequencies lower than physiological. Moreover, the samples with the cluster integration method turned out to be much more resistant (100 % in the cluster method and 25 % in the infusion of isolated cells] to sodium channel inhibitor, lidocaine. The efficiency of integration depends on the conditions of cell growth, which is discussed in more detail in the results of the study. The obtained results are applicable in the development of a biological pacemaker.

Genetic analysis plays an important role in diagnostics of cardiovascular diseases. One of the diseases is long QT syndrome that results in an increased risk of ventricular tachycardia and sudden cardiac death. The syndrome may be caused by mutations in genes responsible for cardiomyocyte ionic channel functioning. The aim of this study is to examine genetics of long QT syndrome. Genetic analysis of 16 patients with long QT syndrome or suspicion of the syndrome was carried out. Long QT syndrome causing mutations, p.Ala178Pro, p.Val254Met, p.Gly325Arg in KCNQ1 and p.Thr613Met in KCNH2, and a long QT syndrome-associated polymorphism, p.Asp85Asn in KCNE1, were found in five patients. Family analysis of p.Ala178Pro and p.Val254Met mutations in KCNQ1 revealed the mutations carriers that had not demonstrated any syndrome manifestations before. In addition, a mutation, p.Gly604Ala in KCNH2, was found. The mutation has not been previously described and its role in long QT syndrome needs to be clarified.

Normal blood levels of potassium are critical for maintaining normal heart electrical rhythm. Both low blood potassium levels (hypokalemia) and high blood potassium levels (hyperkalemia) can lead to abnormal heart rhythms. The aim of the work presented is to study the effect of potassium concentration on the excitation wave in the cardiac tissue. Results have been obtained both in the experimental model, which is a monolayer of neonatal rat cardiomyocytes, and in the modified Korhonen computer model, designed for ventricular rat neonatal cardiomyocytes. The existence of non-sodium excitation waves under a strong hyperkalemia (more than 10 mM K + in the extracellular environment) in the cardiomyocyte monolayer has been found and has also be confirmed by inactivation of sodium channels with a specific channel blocker.

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.

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.

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.

There are risk factors that lead the normal conduction of excitation in the heart into a chaotic one. These factors include hereditary and acquired channelopathies. Many dangerous changes in the work of the heart can be identified using the patient’s electrocardiogram. Such relatively easily detectable changes include the long QT interval syndrome (LQTS). Despite a relatively high prevalence of hereditary LQTS, to which it is necessary to add both hereditary and induced LQTS as well as the ease of detection on the ECG, the mechanism of reentry formation in this syndrome is still un­known. What should be noted is a high variability of the hereditary syndrome and the fact of the connection between the increase in the heart rate and the risk of cardiac arrest. After an electrophysiological study on individual cardiac cells from patients with the LQT syndrome, it became apparent that the search for a mechanism for the transition of the normal heart rhythm to chaotic and fibrillation cannot be limited to recording ion currents in single cells. To solve this problem, we need a model of the behavior of cardiac tissue which reflects the relationship of various factors and the risk of reentry. In order to create an experimental model of LQTS in our work, the iPSC of a pati­ent-specific line from a healthy patient was differentiated into a monolayer of cardiac cells and the parameters of the excitation propagation were studied depending on the stage of differentiation. It was shown that a stable value of the propagation velocity and the response to periodic stimulation in the range of physiological values, are reached after the 30th day of dif­ferentiation.

Building functional and robust scaffolds for engineered biological tissue requires a nanoscale mechanistic understanding of how cells use the scaffold for their growth and development. A vast majority of the scaffolds used for cardiac tissue engineering are based on polymer materials, the matrices of nanofibers. Attempts to load the polymer fibers of the scaffold with additional sophisticated features, such as electrical conductivity and controlled release of the growth factors or other biologically active molecules, as well as trying to match the mechanical features of the scaffold to those of the extracellular matrix, cannot be efficient without a detailed knowledge of how the cells are attached and strategically positioned with respect to the scaffold nanofibers at micro and nanolevel. Studying single cell – single fiber interactions with the aid of confocal laser scanning microscopy (CLSM), scanning probe nanotomography (SPNT), and transmission electron microscopy (TEM), we found that cardiac cells actively interact with substrate nanofibers, but in different ways. While cardiomyocytes often create a remarkable “sheath” structure, enveloping fiber and, thus, substantially increasing contact zone, fibroblasts interact with nanofibers in the locations of focal adhesion clusters mainly without wrapping the fiber. Statements of Significance We found that cardiomyocytes grown on electrospun polymer nanofibers often create a striking “sheath” structure, enveloping fiber with the formation of a very narrow (∼22 nm) membrane gap leading from the fiber to the extracellular space. This wrapping makes the entire fiber surface available for cell attachment. This finding gives a new prospective view on how scaffold nanofibers may interact with growing cells. It may play a significant role in effective design of novel nanofiber scaffolds for tissue engineering concerning mechanical and electrical properties of scaffolds as well as controlled drug release from “smart” biomaterials.

Currently the use of synthetic biodegradable polymers based on polyurethane, polycaprolactone, polylactic and polyglycolic acids structures and their co-polymers is one of the most perspective directions of tissue engineering development. Electrospinning was found as an optimal way to produce nanofibers suitable for building several types of biomaterial scaffolds that are used in cell therapy. This technology allows creating a stable biodegradable and highly biocompatible matrix. In this study we investigate the viability of cardiac fibroblasts cultivated on polymeric nanofibrous scaffolds in vitro and in vivo after implantation in the myocardium of an experimental animal. Polymeric nanofibers were produced on an electrospinning unit. Prepared matrixes were vitalized with cell cultures, received from atriums of several mini-pigs. Cell viability was estimated by the use of XTT based colorimetric assay. Two groups of mini-pigs were selected for this research. The first group underwent a procedure of intramyocardial implantation of a matrix, grown with cardiac cell culture. In the second group a clear polymeric matrix was implanted. Seven days after the procedure animals were sacrificed and fragments of myocardium containing implants were harvested. Frozen sections were prepared immediately, then a standard histological analysis and immunofluorescent staining were performed. Current results can be significant for further development of polymeric scaffolds and for research of biophysical and electrophysiological features of cardiac cell cultures, what will help to expand the abilities of contemporary regenerative medicine and may become a standard of autological biological therapy.

The mechanism of the low voltage defibrillation is based on the drift of the spiral wave induced by a high frequency wave train. In the process, it is first necessary to unpin the wave from the stabilizing obstacle. We study the conditions of unpinning of a rotating wave anchored to the defect by posing the main accent on the boundary conditions of it. The computer simulations performed using the Korhonen model showed that the fluxes through the border of the defect in the cardiac tissue can significantly modify the excitation pattern, and the working frequency gap for the unpinning of reentry waves could be substantially reduced, making overdrive pacing procedure less effective or practically inapplicable.

The work presents a mechanism of spiral wave initiation due to the specific boundary conditions on a border of cardiac tissue defect. There are known scenarios when anatomical or functional defects in cardiac tissue may provoke the spiral wave origination, including unidirectional blockage while passing through the narrow gates, bent over critical curvature wave fronts, inhomogeneous recovery of the tissue, etc. We show a new scenario of spiral wave breakup on a small defect, which is unexcitable but permeable for ionic currents supporting the excitation wave. It was believed that such defects stabilize the rotating wave; however, as shown, instead of stabilizing it leads to the spiral breakup and subsequent multiplication of the rotating waves.

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 μM–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 < 0.05). 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 to 30 μM) at which the probability of re-entry increased.

Substances that can be used as photosensitizers for cardiac tissue are very helpful in modeling various excitation patterns in a cardiac tissue culture and may have prospective use in the temporary and permanent ablation of unwanted excitation sources in the heart. The aim of the present work is to study the effect of stilbene derivative c-TAB (2- {4- [(E) -2- (4-ethoxyphenyl) vinyl] phenoxy} ethyl) trimethylammonium bromide) on the cardiomyocyte layers and voltage-gated ion channels in cardiac cells. C-TAB is a structural analog to AzoTAB, reported previously as a photoswitch for cardiac and neural cells, in which the azobenzene moiety is replaced by a stilbene grouping. Such a replacement makes c-TAB less toxic to living cells. c-TAB has been shown to successfully inhibit excitation in cardiac cells in both trans- and cis- forms. The excitation inhibition of cardiac cells under c-TAB is reversible and can be overturned easily by washing out the c-TAB; however, not by light illumination. The irradiation of cardiac cells with near-UV, when the trans- form of c-TAB is applied, changes reversible inhibition to a permanent one that cannot be overturned by a washout.

In vitro screening for potential side effects of drugs on human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is a cutting-edge technology in pharmaceutical industry. International groups are currently considering using iPSC-CM as a part of comprehensive battery for an accurate and complex mechanistic-based assessment of the proarrhythmic potential of drugs. Despite iPSC-CMs expression and phenotype differences from mature adult CMs screening for drug-induced prolonged QT interval is now routinely carried and also recommended by ICH. The revelation of the mechanism of how the elongation of the QT interval is associated with the occurrence of an arrhythmia should extend the prospects of screening. To address this problem, a comprehensive tissue-based test for arrhythmogenicity is needed. Induced pluripotent stem (iPS) cells from a healthy individual were differentiated into a CM monolayer that was identified by immunocytochemistry and the patch-clamp technique also considering of the potential impact of the developing phenotype of the iPSC-CMs. To study the occurrence of reentry as a precursor to arrhythmias, a standard obstacle was created in the cell layer. With the aid of optical mapping, the measure of arrhythmogenicity was determined, as defined by the probability of a reentry occurrence for the particular frequency of stimulation. A change in the potassium current corresponding to LQTS type 2 at frequencies matching high heart rates was demonstrated visually and quantitatively. Also, the efficiency of this method for quantifying both the effectiveness and ineffectiveness of drugs for a particular donor and for determining the donor's cardiovascular disease risk zone was tested.

Termination of rotating (spiral) waves or reentry is crucial when fighting with the most dangerous cardiac tachyarrhythmia. To increase the efficiency of the antiarrhythmic drugs as well as finding new prospective ones it is decisive to know the mechanisms how they act and influence the reentry dynamics. The most popular view on the mode of action of the contemporary antiarrhythmic drugs is that they increase the core of the rotating wave (reentry) to that extent that it is not enough space in the real heart for the reentry to exist. Since the excitation in cardiac cells is essentially change of the membrane potential, it relies on the functioning of the membrane ion channels. Thus, membrane ion channels serve as primary targets for the substances, which may serve as antiarrhythmics. At least, the entire group of antiarrhythmics class I (modulating activity of sodium channels) and partially class IV (modulating activity of calcium channels) are believed to destabilize and terminate reentry by decreasing the excitability of cardiac tissue. We developed an experimental model employing cardiac tissue culture and photosensitizer (AzoTAB) to study the process of the rotating wave termination while decreasing the excitability of the tissue. A new scenario of spiral wave cessation was observed: an asymmetric growth of the rotating wave core and subsequent formation of a virtual isthmus, which eventually caused a conduction block and the termination of the reentry.

Electrospun fibrous mats are widely used in the contemporary cardiac tissue engineering as the substrates for growing cardiac cells. The substrate with chaotically oriented nanofibers leads to the growth of cardiac tissue with randomly oriented, but internally morphologically anisotropic clusters or domains. The domain structure affects the stability of the excitation propagation and we studied the stability of the propagating excitation waves versus the average size of the domains and the externally applied excitation rate. In an experimental model based on neonatal rat cardiac tissue monolayers, as well as in the computer simulations, we have found that an increase in domain sizes leads to the decrease in the critical stimulation frequencies, thus evidencing that larger domains are having a higher arrhythmogenic effect.

Propagation of the action potential in the real heart is direction-dependent (anisotropic). We propose two general physical models explaining this anisotropy on the cellular level. The first, “delay” model takes into account the frequency of the cell-cell transitions in different directions of propagation, assuming each transition requires some small time interval. The second model relies on the assumption that the action potential transmits to the next cell only from the area at the pole of the previous cell. We estimated parameters of both models by doing optical mapping and fluorescent staining of cardiac cell samples grown on polymer fiber substrate. Both models gave reasonable estimations, but predicted different behaviors of the anisotropy ratio (ratio of the highest and lowest wave velocities) after addition of the suppressor of sodium channels such as lidocaine. The results of the experiment on lidocaine effect on anisotropy ratio were in favor of the first, “delay” model. Estimated average cell-cell transition delay was 240 ± 80 μs, which is close to the characteristic values of synaptic delay.

Bio-actuated micro-pumps do not need any external power source and pose no risk of electrical or heat shock for the biological materials in lab-on-chip systems. Several different designs of bio-actuated micro-pumps based on the use of the contractile force of cultured cardiomyocites have been proposed earlier. Here we present a novel type of a bio-actuated micro-pump representing a microfluidic channel with a contractile wall. The flow inside the channel is generated by the peristaltic movement of its wall caused by the propagation of an excitation-contraction wave along the channels surface. The directional flow generated by the pump was demonstrated by tracking of polystyrene microspheres, moving in the direction of the propagation of the excitation-contraction wave with an average velocity of 6–8 μm/min. The suggested design of a micro-pump allows the control of pumping direction, which might be useful for targeted delivery of fluids and substances in lab-on-chip systems. Prospects of future development and implementation of this kind of bio-actuated peristaltic pumps are discussed.

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.

Cardiac fibrosis occurs in many forms of heart disease and is considered to be one of the main arrhythmogenic factors. Regions with a high density of fibroblasts are likely to cause blocks of wave propagation that give rise to dangerous cardiac arrhythmias. Therefore, studies of the wave propagation through these regions are very important, yet the precise mechanisms leading to arrhythmia formation in fibrotic cardiac tissue remain poorly understood. Particularly, it is not clear how wave propagation is organized at the cellular level, as experiments show that the regions with a high percentage of fibroblasts (65-75%) are still conducting electrical signals, whereas geometric analysis of randomly distributed conducting and non-conducting cells predicts connectivity loss at 40% at the most (percolation threshold). To address this question, we used a joint in vitro-in silico approach, which combined experiments in neonatal rat cardiac monolayers with morphological and electrophysiological computer simulations. We have shown that the main reason for sustainable wave propagation in highly fibrotic samples is the formation of a branching network of cardiomyocytes. We have successfully reproduced the morphology of conductive pathways in computer modelling, assuming that cardiomyocytes align their cytoskeletons to fuse into cardiac syncytium. The electrophysiological properties of the monolayers, such as conduction velocity, conduction blocks and wave fractionation, were reproduced as well. In a virtual cardiac tissue, we have also examined the wave propagation at the subcellular level, detected wavebreaks formation and its relation to the structure of fibrosis and, thus, analysed the processes leading to the onset of arrhythmias., Author summary Cardiac arrhythmias are one of the major causes of death in the industrialized world. The most dangerous ones are often caused by the blocks of propagation of electrical signals. One of the common factors that contribute to the likelihood of these blocks, is a condition called cardiac fibrosis. In fibrosis, excitable cardiac tissue is partially replaced with the inexcitable and non-conducting connective tissue. The precise mechanisms leading to arrhythmia formation in fibrotic cardiac tissue remain poorly understood. Therefore, it is important to study wave propagation in fibrosis from cellular to tissue level. In this paper, we study tissues with high densities of non-conducting cells in experiments and computer simulations. We have observed a paradoxical ability of the tissue with extremely high portion of non-conducting cells (up to 75%) to conduct electrical signals and contract synchronously, whereas geometric analysis of randomly distributed cells predicted connectivity loss at 40% at the most. To explain this phenomenon, we have studied the patterns that cardiac cells form in the tissue and reproduced their self-organisation in a computer model. Our virtual model also took into account the polygonal shapes of the spreading cells and explained high arrhythmogenicity of fibrotic tissue.

Guided excitation in patterned cardiomyocytes was analysed by extracellular recording of field potentials with a commercial device of multi-electrode array (MEA). In order to define fibronectin patterns on the MEA surface, microcontact printing was performed with a microscope-based alignment system. Cardiomyocytes were harvested from neonatal rats and then cultured on the patterned device. After 3days, cell stripes were formed, allowing guided excitation along each of the cell stripes. Increasing the culture time from 3 to 5days improved the beating repeatability without changing the quality of the electric conduction. More detailed analyses revealed that the excitation propagation along a cell stripe was sensitive to the cell coupling defects which could be modified by adding drugs in the culture medium during the early stage of the pattern formation. Our results suggest that extracellular recordings with patterned MEAs are reliable for quantitative analyses of guided excitation of cardiomyocytes.

Aims Development of a human cell-derived reentrant arrhythmia model is needed for studying the mechanisms of disease and accurate drug response. Methods and results We differentiated human pluripotent stem cells (hPSCs) into cardiomyocytes, and then re-plated them into cell sheets that proved capable of forming electrically coupled assemblies. We monitored the function of these re-plated sheets optically with the Ca2+ sensitive dye Fluo-4, and found that they generated characteristic waves of activity whose velocity and patterns of propagation depended upon the concentration of sodium channel blockers; lidocaine and tetrodotoxin, and also the time after re-plating, as well as the applied stimulation frequency. Importantly, reentrant spiral-wave propagation could be generated in these sheets by applying high-frequency stimulation, particularly when cell-density in the sheets was relatively low. This was because cardiac troponin T-positive cells were more non-homogeneously distributed at low cell densities. Especially in such sheets, we could terminate spiral waves by administering the anti-arrhythmic drugs; nifekalant, E-4031, sotalol, and quinidine. We also found that in these sheets, nifekalant showed a clear dose-dependent increase in the size of the unexcitable ‘cores’ of these induced spiral waves, an important parallel with the treatment for ventricular tachycardia in the clinical situation, which was not shown properly in cardiac-cell sheets derived from dissociated rodent hearts. Conclusions We have succeeded in creating from hPSCs a valuable type of cardiomyocyte sheet that is capable of generating reentrant arrhythmias, and thus is demonstrably useful for screening and testing all sorts of drugs with anti-arrhythmic potential.

Recent development of tissue engineering techniques allows creating and maintaining almost indefinitely networks of excitable cells with desired architecture. We coupled the network of live excitable cardiac cells with a common computer by sensitizing them to light, projecting a light pattern on the layer of cells, and monitoring excitation with the aid of fluorescent probes (optical mapping). As a sensitizing substance we used azobenzene trimethylammonium bromide (AzoTAB). This substance undergoes cis-trans-photoisomerization and trans-isomer of AzoTAB inhibits excitation in the cardiac cells, while cis-isomer does not. AzoTAB-mediated sensitization allows, thus, reversible and dynamic control of the excitation waves through the entire cardiomyocyte network either uniformly, or in a preferred spatial pattern. Technically, it was achieved by coupling a common digital projector with a macroview microscope and using computer graphic software for creating the projected pattern of conducting pathways. This approach allows real time interactive photocontrol of the heart tissue.

This paper presents an in vitro system for cardiac tissue engineering based on cardiomyocytes cultured on electrospun polymethylglutarimide (PMGI) nanofibrous meshes either imprinted on solid substrate or suspended in space. Special care was taken over the ability to control the tissue architecture. The electrospinning process allowed nano-scale diameter PMGI fibers with different positioning density to be collected in a random or in an aligned way that defines the general configuration of the mesh. Micro-imprinted on solid substrate nanofibers guarantee aligned cell growth, when the distance between them is 30 μm or less. Suspended in 3D space, nanofibers define the overall architecture of the tissue, depending on orientation and positioning density of the nanofibers. As a result, cardiac cells proliferated into contractile tissue filaments, open-worked tissue meshes and continuous anisotropic cell sheets. Alignment of the cells was characterized by elongation of the cell shape and orientation of the α-actin filaments supported by the FFT data. The advantage of this method is its ability to maintain both three-dimensionality and structural anisotropy.

Cardiomyocyte tissue culture allows visualization of excitation waves with the aid of potential-sensitive and Ca ++ -sensitive dyes similar to experiments with the real heart tissue, but it provides much lower level of intrinsic complexity. It also offers a tool to create desirable structure of the tissue: geometry, controllable inhomogeneities, gradients, etc. In our study we show the ways to create patterned tissue and experimental examples of the processes of origination of spiral waves in cultured cardiomyocytes. We also show the method to control the excitation waves in the cardiomyocyte tissue by adding the AzoTAB and varying the wavelength and intensity of illumination.

We report the vectorial transport of a material object to a desired location by chemical waves generated in an excitable medium (Belousov−Zhabotinsky reaction). The transportation route is determined by the excitable waves induced at a certain point on the excitable medium. This study realized direct coupling between a chemical reaction and vectorial motion; such transduction of energy is generally found in biological motility but has been difficult to achieve in artificial model systems. We discuss the future possible application of these findings to the fabrication of a chemical machine that works under the self-management of motion in response to external stimuli.

In the present study, we examined the ability of the recombinant spidroin to serve as a substrate for the cardiac tissue engineering. For this purpose, isolated neonatal rat cardiomyocytes were seeded on the electrospun spidroin fiber matrices and cultured to form the confluent cardiac monolayers. Besides the adhesion assay and immunostaining analysis, we tested the ability of the cultured cardiomyocytes to form a functional cardiac syncytium by studying excitation propagation in the cultured tissue with the aid of optical mapping. It was demonstrated that recombinant spidroin fiber meshes are directly suitable for the adherence and growth of the cardiomyocytes without additional coating with the attachment factors, such as fibronectin.

Waveblock formation is the main cause of reentry. We have performed a comprehensive numerical modeling study of block formation due to anisotropy in Ten Tusscher and Panfilov (2006) ionic model for human ventricular tissue. We have examined the border between different areas of myocardial fiber alignment and have shown that blockage can occur for a wave traveling from a transverse fiber area to a longitudinal one. Such blockage occurs for reasonable values of the anisotropy ratio (AR): from 2.4 to 6.2 with respect to propagation velocities. This critical AR decreases by the suppression of I Na and I Ca, slightly decreases by the suppression of I Kr and I Ks, and substantially increases by the suppression of I K1. Hyperkalemia affects the block formation in a complex, biphasic way. We provide examples of reentry formation due to the studied effects and have concluded that the suppression of I K1 should be the most effective way to prevent waveblock at the areas of abrupt change in anisotropy.

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.

The dissolution and corrosion of iron and certain types of steel can involve the propagation of electrochemical waves. Low-carbon steel plates covered by nitric acid solutions self-organize pseudo-two-dimensional wave patterns which share many features with reaction-diffusion waves in excitable systems. Here, we investigate the dynamics of these electrodissolution waves in the presence of geometrical constraints. A simple procedure for the preparation of insulating, millimeter-scale obstacles is presented, which allows the controlled, local protection of the metal surface. This method is used in the measurement of the material consumption rate per wave [(850 ( 50) nm/wave or 6-7 g/(m 2 wave)] as well as for the study of front propagation through narrow gaps and long channels. In channels, the wave velocity decreases with decreasing width from 10 to 4 mm/s. Propagation failures occur within the channel if its width is smaller than 0.2 -0.4 mm. These results indicate that the obstacles have sink-like boundaries. For the estimation of the critical nucleation size in this system, we prepared short and narrow gaps that induce wave blockage for openings smaller than 0.65 ( 0.1 mm.

The spatio–temporal dynamics of dissolution patterns on low-carbon steel wires submerged in nitric acid solution are studied in an electrolytic cell by means of optical detection. The cathodic steel wire can segregate into different macroscopic domains. With increasing distance from the external anode, these domains are characterized by constant dissolution, rapidly pulsating oscillations, excitable behavior, and constant passivity. For nitric acid concentrations between 10.5 M and 12.0 M, the fast oscillations trigger traveling waves that experience propagation failures far from the anode. The rhythm of successful wave generation is typically slower and less regular than the rhythm of the rapid oscillations.

Two-dimensional wave patterns have been observed on corroding steel plates. A thin layer of oxygen-containingnitric acid covering the surface of the low-carbon steel induces the corrosion process. The spectrum of theobserved patterns includes propagating pulses, rotating spirals, and serpentine structures. The propagationvelocity is on the order of millimeters per second and increases with acidity. Waves can be observed with thenaked eye as traveling brownish bands on the passivated grayish steel. Spiral wave tips deposit black linesonto the surface, documenting the tip trajectory and influencing the propagation of subsequent spirals. Asimple chemical model for the mechanism of pattern formation is suggested.

We examined chemical oscillations in the Belousov−Zhabotinsky (BZ) reaction with spherical ion-exchange resin beads (radius: 0.1−0.4 mm) loaded with a cationic catalyst, ferroin, in which no traveling wave appears and the oscillation is synchronized over individual beads. The period of the oscillation increases with a decrease in the size of the beads. Based on a theoretical consideration of the effect of the diffusion of active species through the surface of the bead reactor into the bulk aqueous solution, the following relationship was deduced: f/[H2SO4]γ ∝ (1 − η/R) where f is the frequency (Hz) of the oscillation, R is the radius (mm) of the beads, [H2SO4] is the concentration (mol/dm3) of sulfuric acid, andγ and η are constants. This relationship was consistent with experimental results with values of γ = 1.4 and η = 7.4 × 10-4.

A chemical excitable medium was constructed where the velocities of wave propagation to the left and to the right are different. It consists of a network of the “chemical diode” elements, with a gap 150 mm between the “presynaptic” and “postsynaptic” parts. With this gap, a chemical diode conducts excitation in both directions, but with different delays. The network was created by a new method: it was printed by a computer printer on a polysufone membrane preloaded with the catalyst of the reaction. Propagation of plane waves, circular waves and spiral rotating waves was studied. It was found that in an asymmetric medium: (1) Excitation wave length (“wave thickness”) is different for waves propagating to the left and to the right. (2) Target patterns are not of circular form. (3) Spiral waves are not stationary in this medium, they drift with constant velocity. The component C ∥ of the drift is determined by the asymmetry of the medium, the component C ⊥ is controlled by both the asymmetry and the chirality of the spiral.

The tunneling of chemical waves through the gaps of different geometrical shapes was studied using a new experimental model based on the polysulphone membranes with the fixed catalyst. Period doubling of the wave train, formation of the wave breaks on the split in polysulphone membrane, drift of the spiral wave along the widening split were observed. The effects were confirmed and studied in details using computer simulation.

A simple experimental model on the basis of the Belousov−Zhabotinsky (BZ) reaction was produced for the study of wave patterns in a spatially growing excitable medium. A drop of sulfuric acid, placed onto a millipore filter soaked with low-acid BZ solution, expanded with time and made the system excitable. New patterns of flower-like propagation were observed at the frontier of a growing excitable spot. Initially plain circular wave fronts, when expanding, changed to a crown of petal-like propagating fragments of excitation waves. The overall symmetry of the system spontaneously changed with time.

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.

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.