Biomimetic Cardiac Tissue Models for In Vitro Arrhythmia Studies.

Cardiac arrhythmias are a major cause of cardiovascular mortality worldwide. Many arrhythmias are caused by reentry, a phenomenon where excitation waves circulate in the heart. Optical mapping techniques have revealed the role of reentry in arrhythmia initiation and fibrillation transition, but the underlying biophysical mechanisms are still difficult to investigate in intact hearts. Tissue engineering models of cardiac tissue can mimic the structure and function of native cardiac tissue and enable interactive observation of reentry formation and wave propagation. This review will present various approaches to constructing cardiac tissue models for reentry studies, using the authors' work as examples. The review will highlight the evolution of tissue engineering designs based on different substrates, cell types, and structural parameters. A new approach using polymer materials and cellular reprogramming to create biomimetic cardiac tissues will be introduced. The review will also show how computational modeling of cardiac tissue can complement experimental data and how such models can be applied in the biomimetics of cardiac tissue.

Novel Molecular Vehicle-Based Approach for Cardiac Cell Transplantation Leads to Rapid Electromechanical Graft-Host Coupling.

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.

Cellular electrophysiological effects of botulinum toxin A on neonatal rat cardiomyocytes and on cardiomyocytes derived from human-induced pluripotent stem cells.

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.

Polymer Kernels as Compact Carriers for Suspended Cardiomyocytes.

Induced pluripotent stem cells (iPSCs) constitute a potential source of patient-specific human cardiomyocytes for a cardiac cell replacement therapy via intramyocardial injections, providing a major benefit over other cell sources in terms of immune rejection. However, intramyocardial injection of the cardiomyocytes has substantial challenges related to cell survival and electrophysiological coupling with recipient tissue. Current methods of manipulating cell suspensions do not allow one to control the processes of adhesion of injected cells to the tissue and electrophysiological coupling with surrounding cells. In this article, we documented the possibility of influencing these processes using polymer kernels: biocompatible fiber fragments of subcellular size that can be adsorbed to a cell, thereby creating the minimum necessary adhesion foci to shape the cell and provide support for the organization of the cytoskeleton and the contractile apparatus prior to adhesion to the recipient tissue. Using optical excitation markers, the restoration of the excitability of cardiomyocytes in suspension upon adsorption of polymer kernels was shown. It increased the likelihood of the formation of a stable electrophysiological coupling in vitro. The obtained results may be considered as a proof of concept that the stochastic engraftment process of injected suspension cells can be controlled by smart biomaterials.

Muscular Thin Films for Label-Free Mapping of Excitation Propagation in Cardiac Tissue.

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.

Self-organization of conducting pathways explains electrical wave propagation in cardiac tissues with high fraction of non-conducting cells.

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.

Stilbene derivative as a photosensitive compound to control the excitability of neonatal rat cardiomyocytes.

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.

Arrhythmogenicity Test Based on a Human-Induced Pluripotent Stem Cell (iPSC)-Derived Cardiomyocyte Layer.

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.

High resolution 3D microscopy study of cardiomyocytes on polymer scaffold nanofibers reveals formation of unusual sheathed structure.

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.

Biocontractile microfluidic channels for peristaltic pumping.

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.

Virtual cardiac monolayers for electrical wave propagation.

The complex structure of cardiac tissue is considered to be one of the main determinants of an arrhythmogenic substrate. This study is aimed at developing the first mathematical model to describe the formation of cardiac tissue, using a joint in silico-in vitro approach. First, we performed experiments under various conditions to carefully characterise the morphology of cardiac tissue in a culture of neonatal rat ventricular cells. We considered two cell types, namely, cardiomyocytes and fibroblasts. Next, we proposed a mathematical model, based on the Glazier-Graner-Hogeweg model, which is widely used in tissue growth studies. The resultant tissue morphology was coupled to the detailed electrophysiological Korhonen-Majumder model for neonatal rat ventricular cardiomyocytes, in order to study wave propagation. The simulated waves had the same anisotropy ratio and wavefront complexity as those in the experiment. Thus, we conclude that our approach allows us to reproduce the morphological and physiological properties of cardiac tissue.

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes.

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.

Conditions for Waveblock Due to Anisotropy in a Model of Human Ventricular Tissue.

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 INa and ICa, slightly decreases by the suppression of IKr and IKs, and substantially increases by the suppression of IK1. 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 IK1 should be the most effective way to prevent waveblock at the areas of abrupt change in anisotropy.

Functional analysis of the engineered cardiac tissue grown on recombinant spidroin fiber meshes.

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.

Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets.

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 Ca(2+) 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.

Photo-control of excitation waves in cardiomyocyte tissue culture.

Azobenzene photoswitches were recently reported to control the activity of neural cells and heart beat in leeches. Here, we report photocontrol of excitation of cultured cardiomyocytes that have been made light sensitive by using the addition of azobenzene trimethylammonium bromide (AzoTAB). The trans-isomer of AzoTAB reversibly suppresses spontaneous activity and propagation of excitation waves, whereas the cis-isomer has no detectable effect on the electrical properties of cardiomyocytes. Photoisomerization of AzoTAB was achieved by switching the illumination wavelength, λ, from ~440 nm (trans-isomer) to ~350 nm (cis-isomer). Simultaneous irradiation at two wavelengths with properly chosen intensities allowed for dynamic control of the cis-isomer/trans-isomer ratio and the level of excitability from normal to fully unexcitable. Experiments were conducted by using AzoTAB-treated confluent monolayers of neonatal rat cardiomyocytes. Excitation waves were monitored by using the Ca2+-sensitive fluorescent dye Fluo-4. By projecting two-wavelength illumination patterns onto otherwise uniform cell layers, we were able to create excitable networks with the desired topology, dimensions, and functional properties. The present article discusses potential applications of this technique for the analysis of complex patterns of electrical excitation and cardiac arrhythmias.

Electrospun nanofibers as a tool for architecture control in engineered cardiac 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.

Liberation of a pinned spiral wave by a single stimulus in excitable media.

The unpinning of a spiral wave from an anatomic obstacle by the application of a single stimulus near the core of the rotating wave was studied experimentally in a cell culture of cardiomyocyte monolayers as well as by computer simulations. It is shown that, with suitable positioning and timing, a single stimulus is sufficient for the successful unpinning of a pinned spiral wave. Successful unpinning is achieved when two conditions are fulfilled: (1) The stimulus is delivered in the vulnerable window of the rotating wave, and (2) the stimulus is delivered in a spatial zone in proximity to the obstacle, where the shape of the zone is defined by the phase of the anchored spiral wave. Two different scenarios for successful unpinning are discussed, which are distinguished by the distance to the stimuli applied to the obstacle.

Unpinning of a spiral wave anchored around a circular obstacle by an external wave train: common aspects of a chemical reaction and cardiomyocyte tissue.

It is well known that spiral waves are often stabilized by anchoring to a local heterogeneity ("pinning") and that such pinned waves are rather difficult to eliminate. In the present report, we show that pinned spiral waves can be eliminated through collision with a wave train arriving from the outer region, as confirmed in experiments on the Belousov-Zhabotinsky (BZ) reaction as well as in cardiomyocyte tissue culture. A numerical simulation using the Oregonator, a mathematical model for the BZ reaction, provides the parameter area for successful unpinning. The scenario of unpinning is discussed in terms of the dispersion relation of the wave train by taking into account the curvature effect of the excitation wave.

Eliminating spiral waves pinned to an anatomical obstacle in cardiac myocytes by high-frequency stimuli.

The unpinning of spiral waves by the application of high-frequency wave trains was studied in cultured cardiac myocytes. Successful unpinning was observed when the frequency of the paced waves exceeded a critical level. The unpinning process was analyzed by a numerical simulation with a model of cardiac tissue. The mechanism of unpinning by high-frequency stimuli is discussed in terms of local entrainment failure, through a reduction of the two-dimensional spatial characteristics into one dimension.