Cardiomyocyte Network Research Articles

2P290 創薬スクリーニングのための心筋細胞ネット-ワークにおける空間パターンと集団サイズの重要性(26.計測,ポスター,日本生物物理学会年会第51回(2013年度))

2P290 創薬スクリーニングのための心筋細胞ネット-ワークにおける空間パターンと集団サイズの重要性(26.計測,ポスター,日本生物物理学会年会第51回(2013年度))

Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation.

Quantitative Evaluation of Closed-Loop-Shaped Cardiomyocyte Network by Using Ring-Shaped Electrode

Re-entry of excitation in the heart is one of the abnormal phenomena that causes lethal arrhythmia and is thought to be induced by the looped structure of the excitation conduction pathway. To evaluate the geometrical pattern dependence of electrophysiological results, we fabricated three models of cardiomyocyte networks and compared their beating frequencies (BFs), amplitudes of a depolarization peak, and field potential durations (FPDs). The set of different closed-loop-shaped network models from 3 to 8 mm in length showed the same BFs, amplitudes, and FPDs independent of their loop lengths, whereas the BFs and FPDs of 60 µm small clusters, and the FPDs of the 2 mm open-line-shaped network model were different from those of a closed-loop-shaped network model. These results indicate that the mm order larger size of clusters might create lower BFs, and the closed-loop-shaped model may generate longer FPDs. They also suggest the importance of spatial arrangement control of the cardoimyocyte community for reproducible measurement of electrophysiological properties of cardiomyocytes, especially control of the closed-loop formation, which might change the waveforms of FPDs depending on the difference in the geometry and conduction pathway of the cell network.

Quantitative Evaluation of Closed-Loop-Shaped Cardiomyocyte Network by Using Ring-Shaped Electrode

Quantitative Evaluation of Closed-Loop-Shaped Cardiomyocyte Network by Using Ring-Shaped Electrode

Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

Variable T-Tubule Density and Organization in Rat Atrial Cardiomyocytes

T-tubules are invaginations in the cellular membrane of the myocyte. In ventricular cardiomyocytes initiation of excitation-contraction coupling is highly localized near the t-tubules, which span the cell in a predominantly transverse orientation. While t-tubules have traditionally been thought to be virtually absent from atrial cells, recent data indicate that a fairly substantial network is present in the atria of large mammals. We presently compared the t-tubule network of atrial and ventricular cardiomyocytes in 6 week-old Wistar rats. Atrial myocytes were isolated by dicing and agitating tissue in the presence of collagenase, and ventricular cells were isolated by Langendorf perfusion. Cell membrane and t-tubules were stained with di-8-ANEPPS and imaged by confocal microscopy. 100% of ventricular and 37% of atrial cardiomyocytes possessed a measurable t-tubular density (defined as occupying >5% of the cytosolic lumen). In the tubulated atrial cells, t-tubular density was similar to that observed in ventricular myocytes (% cross-sectional area = 16% in atrial vs. 17% in ventricular cardiomyocytes, P=0.76). Surprisingly, 10% of atrial myocytes exhibited an organized, largely transverse structure. Power spectral analysis revealed that this organization was somewhat less homogeneous than that observed in ventricular cells, as atrial myocytes exhibited a higher proportion of longitudinal elements (ratio of peak power to between-peak power = 3.15 in atrial vs. 5.13 in ventricular cells, P=0.02). Neither density nor organization of t-tubules was correlated to atrial cardiomyocyte size. Thus, in contrast to several previous reports, a significant proportion of rat atrial cardiomyocytes possess a t-tubule network which is as extensive as that observed in ventricular cells. Furthermore, in a minority of atrial cardiomyocytes (approximately 10%) the t-tubular network is well organized into a predominately transverse structure.

2D1510 インビトロ心毒性予測のための心筋細胞ネットワークの時間的細胞外電位ゆらぎと空間的伝達速度ゆらぎの評価(計測,口頭発表,日本生物物理学会第50回年会(2012年度))

2D1510 インビトロ心毒性予測のための心筋細胞ネットワークの時間的細胞外電位ゆらぎと空間的伝達速度ゆらぎの評価(計測,口頭発表,日本生物物理学会第50回年会(2012年度))

3PS022 リング状微小電極上の心筋細胞の環状細胞ネットワークを用いた心毒性検査技術(日本生物物理学会第50回年会(2012年度))

3PS022 リング状微小電極上の心筋細胞の環状細胞ネットワークを用いた心毒性検査技術(日本生物物理学会第50回年会(2012年度))

Digital photocontrol of the network of live excitable cells

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.

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

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

Proteome reference map and regulation network of neonatal rat cardiomyocyte

To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data.

Orientation and Community Size Dependences of Pulsatile Electrical Field Stimulation on Lined-Up and Rod-Shaped Single Cardiomyocytes

We have examined the orientation dependence of minimum electric field intensity for the stimulation of cardiomyocytes, which were cultivated in agarose chambers, using a lined-up cardiomyocyte network with different numbers of cells and orientations. When the cell network was arranged parallel to the electric field, the required minimum electric field intensity decreased to one-fourth as cell number increased, whereas that of the cell network arranged orthogonal to the electrical field did not decrease and was independent of cell number. The required electrical field intensity of the 100 µm rod-shaped single cardiomyocyte in a microchamber arranged parallel to the electric field was also 40% lower than that of the cell network arranged orthogonal to the electric field. The results indicate that the gradient of the electric field potential between two ends of the cell network or rod-shaped single cell is important for their excitation.

Quasi-In vivo Heart Electrocardiogram Measurement of ST Period Using Convolution of Cell Network Extracellular Field Potential Propagation in Lined-Up Cardiomyocyte Cell-Network Circuit

A model for the quasi-in vivo heart electrocardiogram (ECG) measurement of the ST period has been developed. As the part of ECG data at the ST period is the convolution of the extracellular field potentials (FPs) of cardiomyocytes in a ventricle, we have fabricated a lined-up cardiomyocyte cell-network on a lined-up microelectrode array and a circular microelectrode in an agarose microchamber, and measured the convoluted FPs. When the ventricular tachyarrhythmias of beating occurred in the cardiomyocyte network, the convoluted FP profile showed similar arrhythmia ECG-like profiles, indicating the convoluted FPs of the in vitro cell network include both the depolarization data and the propagation manner of beating in the heart.

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

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

Mitochondrial Interactosome in Energy Metabolism in Healthy and Cancer Cells

In this study we investigated intracellular distribution of β-tubulin isotypes, expression of mitochondrial creatine kinase (MtCK) and their roles in Mitochondrial Interactosome (supercomplex formed by ATP synthasom, ANT, MtCK, VDAC-tubulin and other VDAC-linker proteins) in adult rat cardiomyocytes, in cancerous HL-1 cells of cardiac phenotype and several other cancer cells lines. Immunofluorescent and western blot analysis of adult cardiac muscle cells revealed the presence of all β-tubulin isotypes with the very regular localization of tubulin II(β2) corresponding to mitochondrial “crystal like” pattern. Antibodies against tubulin IV(β4) revealed characteristic staining of microtubular network. Polymerized transversal lines of tubulin III(β3) matched with Z-lines (alpha-actinin) fixing, probably, microtubular network to sarcomer structure. Tubulin I(β5) distribution was diffusely spotted and fragmentary polymerized. In cancer cardiac HL-1 and other cancer cell types MI remodelling is characterized by the absence of II(β2). Additionally, these cells do not contain tubulin IV(β4) isotype creating micro-tubular network in adult cardiomyocytes and have diffusely distributed tubulin I(β5) and III(β3) isotypes. Other characteristic of cancer cells was the absence of both sarcomeric and ubiquitous mitochondrial isoform of creatine kinase (MtCK). In cardiac cells respiration is controlled by creatine via activation of MtCK within MI. Knowing that the extremely low apparent Km for free ADP in cancer cells as well as in isolated mitochondria is associated with the lack of tubulin II(β2) isotype, we can assume that tubulin II(β2) can specifically control the voltage dependent anion channel (VDAC) selective permeability resulting in adenine nucleotides micro-compartmentation within the MI. The absence of II(β2) tubulin isotype and MtCK isoforms induce significant alterations of phenotype and regulatory mechanisms of cancer cells energy metabolism. By these mechanisms, all mitochondrial ATP can be captured to maintain activated glycolysis and lactate production, known as Warburg effect.

2J1448 Quasi-in vivo heart electrocardiogram measurement using convolution of field potential propagation in cardiomyocyte network circuit(Measurements, Miscellaneous topics,The 48th Annual Meeting of the Biophysical Society of Japan)

2J1448 Quasi-in vivo heart electrocardiogram measurement using convolution of field potential propagation in cardiomyocyte network circuit(Measurements, Miscellaneous topics,The 48th Annual Meeting of the Biophysical Society of Japan)

Microtopographical effects of natural scaffolding on cardiomyocyte function and arrhythmogenesis

A natural myocardial patch for heart regeneration derived from porcine urinary bladder matrix (UBM) was previously reported to outperform synthetic materials (Dacron and expanded polytetrafluoroethylene (ePTFE)) used in current surgical treatments. UBM, an extracellular matrix prepared from urinary bladder, has intricate three-dimensional architecture with two distinct sides: the luminal side with a smoother surface relief; and the abluminal side with a fine mesh of nano- and microfibers. This study tested the ability of this natural scaffold to support functional cardiomyocyte networks, and probed how the local microtopography and composition of the two sides affects cell function. Cardiomyocytes isolated from neonatal rats were seeded in vitro to form cardiac tissue onto luminal (L) or abluminal (Ab) UBM. Immunocytochemistry of contractile cardiac proteins demonstrated growth of cardiomyocyte networks with mature morphology on either side of UBM, but greater cell compactness was seen in L. Fluorescence-based imaging techniques were used to measure dynamic changes in intracellular calcium concentration upon electrical stimulation of L and Ab-grown cells. Functional differences in cardiac tissue grown on the two sides manifested themselves in faster calcium recovery (p<0.04) and greater hysteresis (difference in response to increasing and decreasing pacing rates) for L vs Ab side (p<0.03). These results suggest that surface differences may be leveraged to engineer the desired cardiomyocyte responses and highlight the potential of natural scaffolds for fostering heart repair.

Community Effect on Drug Sensitivity of Cardiomyocytes Controlled Spatial Patterns by using On-Chip MEA System

In cardiomyocyte network, network size and spatial arrangement of different cellular-type are important factors for stabilization of beating rhythms. To study the community effect of the geometric factor and community size, we tried to develop cultivation chambers in a various size and geometric patterns using the agarose micro-processing technique combined with the multi-electrode array (MEA) measurement system (we call it On-Chip MEA system), and each of extra-cellular signal of mouse embryonic cardiomyocytes to tachyrhythmia inducing drug response was recorded simultaneously.Firstly, to evaluate the effect of community size on the sensitivity to drug, we tried to build the square sheet type chambers having small, medium and large area (about 104, 105, and 106 um2, respectively). Next, we tried to construct circuit type chambers with loop structure (circuit length of 2um, 8.2um) compared with sheet type geometry.As a result, we observed different response to drug among community size and geometric pattern. These results imply that sensitivity to the drug depends on spatial patterns. In this meeting, difference of drug sensitive event (e.g. tachyrhthmia, cardiac arrest etc), we report in detail about quantitative parameters: Beating Rate (BR), Field Potential Duration (FPD), Short Term Variability (STV) of them etc, which will be help for understanding community effect.

Community Effect to the External Electrical Stimulation on Cardiomyocytes by using On-Chip MEA System

In the cardiomyocyte network, network size and spatial arrangement of different cellular-type are important factors for reducing the fluctuation of the beating rhythms. To study the community effect on the cell network, experimental conditions need to control the community size and to construct the network in a stepwise manner. Therefore, we tried to develop a various size of cultivation chamber using the agarose micro-chamber system combined with the multi-electrode array (MEA) measurement system (we call it On-Chip MEA system).

Electrical Behavior At the Transverse-Axial Tubular Membrane of Hypertrophic Cardiomyocytes in Situ

Cardiac hypertrophy is associated with restructuring of the transverse-axial tubular (TAT) system. It is unknown whether such changes are accompanied by altered electrical behavior at the transverse-axial tubules. Here, we used confocal microscopy and the voltage-sensitive dye ANNINE-6plus to visualize the TAT network and to resolve action potentials at the surface and TAT membrane of in situ left ventricular cardiomyocytes within normal and hypertrophic mouse hearts. Transgenic (TG) hearts overexpressing Gαq and non-transgenic (NTG) hearts were perfused in Langendorff-mode during image acquisition. Cardiomyocytes in hypertrophic TG hearts exhibited patchy loss and gain, respectively, of transverse and axial elements. Action potentials were recorded at the TAT membrane within two adjacent cardiomyocytes (denoted T1 and T2) and at the end-to-end junctions (E) between them. Action potential onset occurred synchronously within the TAT network of NTG and TG cardiomyocytes. Whereas the repolarization time course was similar between the three recording sites in NTG or TG hearts (Figure A), overall repolarization was prolonged in TG cardiomyocytes (Figure B). Thus, electrical coupling between the surface and TAT membrane was maintained despite pronounced TAT membrane restructuring in this model of cardiac hypertrophy.View Large Image | View Hi-Res Image | Download PowerPoint Slide