Cardiac Cell Monolayers Research Articles

Versatile electrical stimulator for cardiac tissue engineering-Investigation of charge-balanced monophasic and biphasic electrical stimulations.

The application of biomimetic physical stimuli replicating the in vivo dynamic microenvironment is crucial for the in vitro development of functional cardiac tissues. In particular, pulsed electrical stimulation (ES) has been shown to improve the functional properties of in vitro cultured cardiomyocytes. However, commercially available electrical stimulators are expensive and cumbersome devices while customized solutions often allow limited parameter tunability, constraining the investigation of different ES protocols. The goal of this study was to develop a versatile compact electrical stimulator (ELETTRA) for biomimetic cardiac tissue engineering approaches, designed for delivering controlled parallelizable ES at a competitive cost. ELETTRA is based on an open-source micro-controller running custom software and is combinable with different cell/tissue culture set-ups, allowing simultaneously testing different ES patterns on multiple samples. In particular, customized culture chambers were appositely designed and manufactured for investigating the influence of monophasic and biphasic pulsed ES on cardiac cell monolayers. Finite element analysis was performed for characterizing the spatial distributions of the electrical field and the current density within the culture chamber. Performance tests confirmed the accuracy, compliance, and reliability of the ES parameters delivered by ELETTRA. Biological tests were performed on neonatal rat cardiac cells, electrically stimulated for 4days, by comparing, for the first time, the monophasic waveform (electric field = 5V/cm) to biphasic waveforms by matching either the absolute value of the electric field variation (biphasic ES at ±2.5V/cm) or the total delivered charge (biphasic ES at ±5V/cm). Findings suggested that monophasic ES at 5V/cm and, particularly, charge-balanced biphasic ES at ±5V/cm were effective in enhancing electrical functionality of stimulated cardiac cells and in promoting synchronous contraction.

Paradoxical Onset of Arrhythmic Waves from Depolarized Areas in Cardiac Tissue Due to Curvature-Dependent Instability.

The generation of abnormal excitations in pathological regions of the heart is a main trigger for lethal cardiac arrhythmias. Such abnormal excitations, also called ectopic activity, often arise from areas with local tissue heterogeneity or damage accompanied by localized depolarization. Finding the conditions that lead to ectopy is important to understand the basic biophysical principles underlying arrhythmia initiation and might further refine clinical procedures. In this study, we are the first to address the question of how geometry of the abnormal region affects the onset of ectopy using a combination of experimental, in silico, and theoretical approaches. We paradoxically find that, for any studied geometry of the depolarized region in optogenetically modified monolayers of cardiac cells, primary ectopic excitation originates at areas of maximal curvature of the boundary, where the stimulating electrotonic currents are minimal. It contradicts the standard critical nucleation theory applied to nonlinear waves in reaction-diffusion systems, where a higher stimulus is expected to produce excitation more easily. Our in silico studies reveal that the nonconventional ectopic activity is caused by an oscillatory instability at the boundary of the damaged region, the occurrence of which depends on the curvature of that boundary. The onset of this instability is confirmed using the Schrödinger equation methodology proposed by Rinzel and Keener [SIAM J. Appl. Math. 43, 907 (1983)]. Overall, we show distinctively novel insight into how the geometry of a heterogeneous cardiac region determines ectopic activity, which can be used in the future to predict the conditions that can trigger cardiac arrhythmias.

The study of the functionality of cardiomyocytes obtained from induced pluripotent stem cells for the modeling of cardiac arrhythmias based on long QT syndrome

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.

In silico study of multicellular automaticity of heterogeneous cardiac cell monolayers: Effects of automaticity strength and structural linear anisotropy.

The biological pacemaker approach is an alternative to cardiac electronic pacemakers. Its main objective is to create pacemaking activity from added or modified distribution of spontaneous cells in the myocardium. This paper aims to assess how automaticity strength of pacemaker cells (i.e. their ability to maintain robust spontaneous activity with fast rate and to drive neighboring quiescent cells) and structural linear anisotropy, combined with density and spatial distribution of pacemaker cells, may affect the macroscopic behavior of the biological pacemaker. A stochastic algorithm was used to randomly distribute pacemaker cells, with various densities and spatial distributions, in a semi-continuous mathematical model. Simulations of the model showed that stronger automaticity allows onset of spontaneous activity for lower densities and more homogeneous spatial distributions, displayed more central foci, less variability in cycle lengths and synchronization of electrical activation for similar spatial patterns, but more variability in those same variables for dissimilar spatial patterns. Compared to their isotropic counterparts, in silico anisotropic monolayers had less central foci and displayed more variability in cycle lengths and synchronization of electrical activation for both similar and dissimilar spatial patterns. The present study established a link between microscopic structure and macroscopic behavior of the biological pacemaker, and may provide crucial information for optimized biological pacemaker therapies.

A Cell-Based Framework for Numerical Modeling of Electrical Conduction in Cardiac Tissue

In this paper, we study a mathematical model of cardiac tissue based on explicit representation of individual cells. In this EMI model, the extracellular (E) space, the cell membrane (M) and the intracellular (I) space are represented as separate geometrical domains. This representation introduces modelling flexibility needed for detailed representation of the properties of cardiac cells including their membrane. In particular, we will show that the model allows ion channels to be non-uniformly distributed along the membrane of the cell. Such features are difficult to include in classical homogenized models like the monodomain and bidomain models frequently used in computational analyses of cardiac electrophysiology. The EMI model is solved using a finite difference method (FDM) and two variants of the finite element method (FEM). We compare the three schemes numerically, reporting on CPU-efforts and convergence rates. Finally, we illustrate the distinctive capabilities of the EMI model compared to classical models by simulating monolayers of cardiac cells with heterogeneous distributions of ionic channels along the cell membrane. Because of the detailed representation of every cell, the computational problems that result from using the EMI model are much larger than for the classical homogenized models, and thus represent a computational challenge. However, our numerical simulations indicate that the FDM scheme is optimal in the sense that the computational complexity increases proportionally to the number of cardiac cells in the model. Moreover, we present simulations, based on systems of equations involving ~ 117 million unknowns, representing up to ~ 16000 cells. We conclude that collections of cardiac cells can be simulated using the EMI model, and that the EMI model enable greater modeling flexibility than the classical monodomain and bidomain models.

Entropy Rate Maps of Complex Excitable Dynamics in Cardiac Monolayers

The characterization of spatiotemporal complexity remains a challenging task. This holds in particular for the analysis of data from fluorescence imaging (optical mapping), which allows for the measurement of membrane potential and intracellular calcium at high spatial and temporal resolutions and, therefore, allows for an investigation of cardiac dynamics. Dominant frequency maps and the analysis of phase singularities are frequently used for this type of excitable media. These methods address some important aspects of cardiac dynamics; however, they only consider very specific properties of excitable media. To extend the scope of the analysis, we present a measure based on entropy rates for determining spatiotemporal complexity patterns of excitable media. Simulated data generated by the Aliev–Panfilov model and the cubic Barkley model are used to validate this method. Then, we apply it to optical mapping data from monolayers of cardiac cells from chicken embryos and compare our findings with dominant frequency maps and the analysis of phase singularities. The studies indicate that entropy rate maps provide additional information about local complexity, the origins of wave breakup and the development of patterns governing unstable wave propagation.

Multicellular automaticity of cardiac cell monolayers: effects of density and spatial distribution of pacemaker cells

Self-organization of pacemaker (PM) activity of interconnected elements is important to the general theory of reaction–diffusion systems as well as for applications such as PM activity in cardiac tissue to initiate beating of the heart. Monolayer cultures of neonatal rat ventricular myocytes (NRVMs) are often used as experimental models in studies on cardiac electrophysiology. These monolayers exhibit automaticity (spontaneous activation) of their electrical activity. At low plated density, cells usually show a heterogeneous population consisting of PM and quiescent excitable cells (QECs). It is therefore highly probable that monolayers of NRVMs consist of a heterogeneous network of the two cell types. However, the effects of density and spatial distribution of the PM cells on spontaneous activity of monolayers remain unknown. Thus, a simple stochastic pattern formation algorithm was implemented to distribute PM and QECs in a binary-like 2D network. A FitzHugh–Nagumo excitable medium was used to simulate electrical spontaneous and propagating activity. Simulations showed a clear nonlinear dependency of spontaneous activity (occurrence and amplitude of spontaneous period) on the spatial patterns of PM cells. In most simulations, the first initiation sites were found to be located near the substrate boundaries. Comparison with experimental data obtained from cardiomyocyte monolayers shows important similarities in the position of initiation site activity. However, limitations in the model that do not reflect the complex beat-to-beat variation found in experiments indicate the need for a more realistic cardiomyocyte representation.

Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells

Interleukin-6 mediates host defense and cell survival mainly through the activation of the transcription factor STAT3 via the glycoprotein gp130, a shared signal-transducing receptor for several IL-6-type cytokines. We have reported that the cardiotrophic parasite Trypanosoma cruzi protects murine cardiomyocytes from apoptosis. In agreement, an intense induction of the anti-apoptotic factor Bcl-2 is found in cardiac fibers during the acute phase of infection, establishing a higher threshold against apoptosis. We report here that inactive cruzipain, the main cysteine protease secreted by the parasite, specifically triggered TLR2 and the subsequent release of IL-6, which acted as an essential anti-apoptotic factor for cardiomyocyte cultures. Although comparable IL-6 levels were found under active cruzipain stimulation, starved cardiac cell monolayers could not be rescued from apoptosis. Moreover, cardiomyocytes treated with active cruzipain completely abrogated the STAT3 phosphorylation and nuclear translocation induced by recombinant IL-6. This inhibition was also observed on splenocytes, but it was reverted when the enzyme was complexed with chagasin, a parasite cysteine protease inhibitor. Furthermore, the inhibition of IL-6-induced p-STAT3 was evidenced in spleen cells stimulated with pre-activated supernatants derived from trypomastigotes. To account for these observations, we found that cruzipain enzymatically cleaved recombinant gp130 ectodomain, and induced the release of membrane-distal N-terminal domain of this receptor on human peripheral blood mononuclear cells. These results demonstrate, for the first time, that the parasite may modify the IL-6-induced response through the modulation of its cysteine protease activity, suggesting that specific inhibitors may help to improve the immune cell activation and cardioprotective effects.

Optical imaging of arrhythmias in the cardiomyocyte monolayer

In recent years, cultured cardiac cell monolayers have become a contemporary experimental preparation for the study of fundamental mechanisms that underlie normal and pathologic electrophysiology at the tissue level. Ion channels and gap junctions in the cardiomyocyte monolayer may be modulated using drugs that suppress or enhance certain channels/junctions, or by genetic silencing or overexpression. The cardiomyocyte monolayer is particularly well suited for studies of functional electrophysiologic properties of mixtures of cardiac and noncardiac cells (eg, myofibroblasts), which otherwise would be difficult to investigate. Optical mapping of monolayers has provided insight into mechanisms that can set the stage for arrhythmias, such as unidirectional conduction block, gap junction uncoupling, ischemia, alternans, and anisotropy, and continues to enhance our understanding of basic electrophysiologic mechanisms.

Collision-Based Spiral Acceleration in Cardiac Media: Roles of Wavefront Curvature and Excitable Gap

We have previously shown in experimental cardiac cell monolayers that rapid point pacing can convert basic functional reentry (single spiral) into a stable multiwave spiral that activates the tissue at an accelerated rate. Here, our goal is to further elucidate the biophysical mechanisms of this rate acceleration without the potential confounding effects of microscopic tissue heterogeneities inherent to experimental preparations. We use computer simulations to show that, similar to experimental observations, single spirals can be converted by point stimuli into stable multiwave spirals. In multiwave spirals, individual waves collide, yielding regions with negative wavefront curvature. When a sufficient excitable gap is present and the negative-curvature regions are close to spiral tips, an electrotonic spread of excitatory currents from these regions propels each colliding spiral to rotate faster than the single spiral, causing an overall rate acceleration. As observed experimentally, the degree of rate acceleration increases with the number of colliding spiral waves. Conversely, if collision sites are far from spiral tips, excitatory currents have no effect on spiral rotation and multiple spirals rotate independently, without rate acceleration. Understanding the mechanisms of spiral rate acceleration may yield new strategies for preventing the transition from monomorphic tachycardia to polymorphic tachycardia and fibrillation.

Effect of Regional Mitochondrial Uncoupling on Electrical Propagation in Cardiac Cell Monolayers

The collapse of mitochondrial inner-membrane potential (ΔΨm) is a key determinant of cell injury and arrhythmogenesis associated with ischemia-reperfusion or oxidative stress. We have previously demonstrated that mitochondrial uncoupling resulting from the activation of energy-dissipating mitochondrial channels can activate sarcolemmal KATP channels, which can profoundly alter cellular electrical excitability. We proposed that these regions could serve as “metabolic sinks” of current that could be a substrate for reentry. Here, we directly test whether local ΔΨm loss influences electrical propagation in monolayers of neonatal rat ventricular myocytes (NRVMs) using a method of local perfusion of a portion of the monolayer with the mitochondrial oxidative phosphorylation uncoupler, FCCP (a protonophore; 1μM). Propagation of the electrical wave through the monolayer was recorded by optical mapping with a 464-element photodiode array using the voltage-sensitive fluorescent dye, di-4-ANEPPS. Using a custom-built perfusion device, a 5mm circular zone in the center of the coverslip (full diameter 2cm) was exposed to FCCP with the remainder perfused with normal Tyrode's buffer, and the monolayer was stimulated from one edge at 1Hz. Upon encountering the metabolic sink, the wave of electrical depolarization was slowed and the amplitude of the action potentials in the FCCP-perfused area decreased significantly. The results indicate that heterogeneous ΔΨm collapse can significantly alter the electrical substrate in a manner that could promote reentry.

Genesis of complex fractionated atrial electrograms in zones of slow conduction: A computer model of microfibrosis

Complex fractionated atrial electrograms are used as potential targets for catheter ablation therapy of atrial fibrillation. Although fibrosis has been associated with the presence of fractionated electrograms, characterizing the substrate through the inspection of electrograms is challenging. This study sought to determine how progression of microfibrosis and slow conduction affect electrogram morphology. A microstructure computer model representing a monolayer of cardiac cells was developed. Slow conduction was induced by: (1) sodium channel blockade, (2) uniform reduction in cell-to-cell coupling, and (3) microfibrosis incorporated as a set of collagenous septa disconnecting transverse coupling. The density (0 to 30%) and length (30 to 945 microm) of these collagenous septa were varied. Unipolar and bipolar electrograms were computed during paced rhythm for a set of electrodes with different tip sizes. The analysis of unipolar electrograms with realistic temporal and spatial filtering showed that increasing the density and length of collagenous septa decreased conduction velocity by up to 75% and increased the amount of fractionation (up to 14 deflections) and asymmetry of the electrograms. In contrast, slow conduction induced by sodium channel blockade or uniformly reduced coupling did not result in electrogram fractionation. When a larger electrode was used, electrogram amplitude was smaller and fractionation increased in a substrate-dependent way. Microscale obstacles cause significant changes to electrogram waveforms. Conduction velocity and electrogram amplitude and degree of fractionation can be used to discriminate the nature of the substrate and characteristics of fibrosis, giving rise to slow conduction.

Targeted Stabilisation Of The RyR2 I-Domain Restores Ca2+ Handling And Intercellular Synchrony In Ouabain-disrupted Cardiac Cell Monolayers

Stabilisation of cardiac ryanodine receptors (RyR2) has emerged as an important approach for normalising Ca2+ cycling dysfunction in cardiac disease. Here, we targeted the I-domain of human RyR2, a region critically involved in RyR2 intramolecular rearrangement following channel activation. The I-domain is a hot-spot for arrhythmia-linked mutations that destabilise the activated channel. Putative functional motifs within the I-domain were mapped using a bioinformatics approach and the synthesised using an in vitro expression system. The efficacy of affinity-purified fragments in normalising Ca2+ handling and intercellular coupling was screened using a cardiac-cell model of ouabain-induced dysynchrony. Under defined experimental conditions, ouabain perturbs normal intracellular Ca2+ cycling and ablates the synchronous intercellular coupling in super-confluent HL-1 monolayers. A 146 amino acid fragment, termed IDB, normalised ouabain-induced Ca2+ dysfunction and resulted in the re-synchronisation of Ca2+ transients across the monolayer. In the same ouabain-disrupted model , bacterially-synthesised IDB increased the extent of intercellular synchrony to levels greater than those measured in naive (non-ouabain treated) spontaneously contractile HL-1 cells. The fortuitous presence of overlapping, truncated recombinant fragments that spanned the entire IDB sequence and co-purified with intact IDB from bacterial culture may have contributed to these effects. Importantly, IDB-mediated normalisation of Ca2+ handling and intercellular synchronisation within the ouabain-treated monolayer extended to distant cell populations that had not been transduced with the recombinant protein. In experiments using cyan-yellow bio-engineered RyR2 (CYBER) probes that report intra-RyR2 conformational rearrangement, we showed that the IDB-mediated stabilisation of the RyR2 channel directly correlated with the normalisation of ouabain-induced Ca2+ dysfunction. Our data provides evidence that a specific epitope-targeting strategy can stabilise RyR2 and that this approach may have remarkable therapeutic utility in normalising channel abnormalities associated with acquired and genetic cardiac disease.

Representation of Collective Electrical Behavior of Cardiac Cell Sheets

The electrocardiogram (ECG) is a measure of the collective electrical behavior of the heart based on body surface measurements. With computational models or tissue preparations, various methods have been used to compute the pseudo-ECG ( pECG) of bipolar and unipolar leads that can be given clinical interpretation. When spatial maps of transmembrane potential ( V m) are available, pECG can be derived from a weighted sum of the spatial gradients of V m. The concept of a lead field can be used to define sensitivity curves for different bipolar and unipolar leads and to determine an effective operating height for the bipolar lead position for a two-dimensional sheet of heart cells. The pseudo-vectorcardiogram ( pVCG) is computed from orthogonal bipolar lead voltages, which are derived in this study from optical voltage maps of cultured monolayers of cardiac cells. Rate and propagation direction for paced activity, rotation frequency for reentrant activity, direction of the common pathway for figure-eight reentry, and transitions from paced activity to reentry can all be distinguished using the pVCG. In contrast, the unipolar pECG does not clearly distinguish among many of the different types of electrical activity. We also show that pECG can be rapidly computed by two geometrically weighted sums of V m, one that is summed over the area of the cell sheet and the other over the perimeter of the cell sheet. Our results are compared with those of an ad hoc difference method used in the past that consists of a simple difference of the sum of transmembrane potentials on one side of a tissue sheet and that of the other.

Corrigendum

CORRIGENDACorrigendumPublished Online:01 Mar 2008https://doi.org/10.1152/ajpheart.zh4-8142-corr.2008Original articleMoreSectionsPDF (33 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Volume 294, January 2008 Volume 63, January 2008 Pages H58–H65: Lin JW, Garber L, Qi YR, Chang MG, Cysyk J, Tung L. “Region of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers.” On p. H58 (title page of the article) and on p. F5 (first page of the Table of Contents for the journal issue), the correct title to the article should read as follows: “Region of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers.”This article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationRelated ArticlesRegion of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers 01 Jan 2008American Journal of Physiology-Heart and Circulatory PhysiologyCited ByNegative Curvature and Control of Excitable Biological Media2 August 2015 More from this issue > Volume 294Issue 3March 2008Pages H1501-H1501 Copyright & PermissionsCopyright © 2008 by the American Physiological Societyhttps://doi.org/10.1152/ajpheart.zh4-8142-corr.2008History Published online 1 March 2008 Published in print 1 March 2008 Metrics

Region of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers

Pathophysiological heterogeneity in cardiac tissue is related to the occurrence of arrhythmias. Of importance are regions of slowed conduction, which have been implicated in the formation of conduction block and reentry. Experimentally, it has been a challenge to produce local heterogeneity in a manner that is both reversible and well controlled. Consequently, we developed a dual-zone superfusion chamber that can dynamically create a small (5 mm) central island of heterogeneity in cultured cardiac cell monolayers. Three different conditions were studied to explore the effect of regionally slowed conduction on wave propagation and reentry: depolarization by elevated extracellular potassium, sodium channel inhibition with lidocaine, and cell-cell decoupling with palmitoleic acid. Using optical mapping of transmembrane voltage, we found that the central region of slowed conduction always served as the core region around which a spiral wave formed and then revolved following a period of rapid pacing. Because of the localized slowing in the core region, we observed experimentally for the first time an S shape of the spiral wave front near its tip. These results indicate that a small region of slowed conduction can play a crucial role in the formation, anchoring, and modulation of reentrant spiral waves.

Spatial Frequency Content in Optical Mapping of Cardiac Cell Monolayers

Fluorescence imaging using voltage-sensitive dyes is an important tool for studying electrical propagation in the heart. Yet, the low amplitude of the voltage-sensitive component in the fluorescence signal and high acquisition rates dictated by the rapid propagation of the excitation wave front make it difficult to achieve recordings with high signal-to-noise ratios. Although spatially and temporally filtering the acquired signals has become de facto one of the key elements of optical mapping, there is no consensus regarding their use. Here we characterize the spatiotemporal spectra of optically recorded action potentials and determine the distortion produced by conical filters of different sizes. On the basis of these findings, we formulate the criteria for rational selection of filter characteristics. We studied the evolution of the spatial spectra of the propagating wave front after epicardial point stimulation of the isolated, perfused right ventricular free wall of the pig heart stained with di-4-ANEPPS. We found that short-wavelength (<3 mm) spectral components represent primarily noise and surface features of the preparation (coronary vessels, fat, and connective tissue). The time domain of the optical action potential spectrum also lacks high-frequency components (>100 Hz). Both findings are consistent with the reported effect of intrinsic blurring caused by light scattering inside the myocardial wall. The absence of high-frequency spectral components allows the use of aggressive low-pass spatial and temporal filters without affecting the optical action potential morphology. We show examples where the signal-to-noise ratio increased up to 150 with <3% distortion. A generalization of our approach to the rational filter selection in various applications is discussed.

Structure-Related Initiation of Reentry by Rapid Pacing in Monolayers of Cardiac Cells

This study examines how a zigzag pattern of conduction, a form of structural heterogeneity frequently found in old or diseased hearts, affects the vulnerability to reentry during rapid pacing. A central rectangular island (8x4 mm) containing a predefined zigzag pattern was created in cultured isotropic monolayers of neonatal rat ventricular myocytes. Impulse propagation was optically mapped from 253 sites using voltage-sensitive dye and was anisotropic within the zigzag island. With increasing interval between neighboring transverse connections (a), relative to the distance between longitudinal strands (b), transverse conduction velocity (CV) decreased to 66+/-6%, 20+/-2%, and 15+/-2% of CV in the surrounding isotropic region, whereas longitudinal CV increased to 102+/-8%, 113+/-12%, and 131+/-23% for a:b ratios of 1:1, 1:5, and 1:9, respectively. During rapid pacing, propagation distal to the island was steered from the side of the island with more transverse connections ("dominant" side) toward the side with fewer connections ("weak" side). Increased asymmetry in the pattern accentuated this effect, and resulted in increased rate-dependent differences in CV on the 2 sides. Consequently, a functional obstacle formed on the weak side, followed by development of single loop reentry. The reentrant wave revolved around a line of block defined by the border of the island. Reentry chirality was determined by the weak side location, and the pacing rate needed to initiate reentry decreased with increased asymmetry in the pattern. In conclusion, reentry is readily induced by rapid pacing in confluent cardiac cell monolayers containing a central and asymmetric island of zigzag conduction.

Shear stress predicts distribution of high strain spots on plaques in human coronary arteries

Although stretch-activated currents have been extensively studied in isolated cells and intact heart in the context of mechanoelectric feedback (MEF) in the heart, quantitative data regarding other mechanical parameters such as pressure, shear, bending, etc, are still lacking at the multicellular level. Cultured cardiac cell monolayers have been used increasingly in the past decade as an in vitro model for the studies of fundamental mechanisms that underlie normal and pathological electrophysiology at the tissue level. Optical mapping makes possible multisite recording and analysis of action potentials and wavefront propagation, suitable for monitoring the electrophysiological activity of the cardiac cell monolayer under a wide variety of controlled mechanical conditions. In this paper, we review methodologies that have been developed or could be used to mechanically perturb cell monolayers, and present some new results on the acute effects of pressure, shear stress and anisotropic strain on cultured neonatal rat ventricular myocyte (NRVM) monolayers.

In Vivo Conditioning of Tissue‐engineered Heart Muscle Improves Contractile Performance

The ability to engineer cardiac tissue in vitro is limited by the absence of a vasculature. In this study we describe an in vivo model which allows neovascularization of engineered cardiac tissue. Three-dimensional cardiac tissue, termed "cardioids," was engineered in vitro from the spontaneous delamination of a confluent monolayer of cardiac cells. Cardioids were sutured onto a support framework and then implanted in a subcutaneous pocket in syngeneic recipient rats. Three weeks after implantation, cardioids were recovered for in vitro force testing and histological evaluation. Staining for hematoxylin and eosin demonstrated the presence of viable cells within explanted cardioids. Immunostaining with von Willebrand factor showed the presence of vascularization. Electron micrographs revealed the presence of large amounts of aligned contractile proteins and a high degree of intercellular connectivity. The peak active force increased from an average value of 57 microN for control cardioids to 447 microN for explanted cardioids. There was also a significant increase in the specific force. There was a significant decrease in the time to peak tension and half relaxation time. Explanted cardioids could be electrically paced at frequencies of 1-5 Hz. Explanted cardioids exhibited a sigmoidal response to calcium and positive chronotropy in response to epinephrine. As the field of cardiac tissue engineering progresses, it becomes desirable to engineer larger diameter tissue equivalents and to induce angiogenesis within tissue constructs. This study describes a relatively simple in vivo model, which promotes the neovascularization of tissue-engineered heart muscle and subsequent improvement in contractile performance.