Search

We examined a simultaneous combined spatiotemporal field potential duration (FPD) and cell-to-cell conduction time (CT) in lined-up shaped human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using an on-chip multielectrode array (MEA) system to evaluate two origins of lethal arrhythmia, repolarization and depolarization. The repolarization index, FPD, was prolonged by E-4031 and astemizole, and shortened by verapamil, flecainide and terfenadine at 10 times higher than therapeutic plasma concentrations of each drug, but it did not change after lidocaine treatment up to 100 μM. CT was increased by astemizol, flecainide, terfenadine, and lidocaine at equivalent concentrations of Nav1.5 IC50, suggesting that CT may be an index of cardiac depolarization because the increase in CT (i.e., decrease in cell-to-cell conduction speed) was relevant to Nav1.5 inhibition. Fluctuations (short-term variability; STV) of FPD and CT, STVFPD and STVCT also discriminated between torsadogenic and non-torsadogenic compounds with significant increases in their fluctuation values, enabling precise prediction of arrhythmogenic risk as potential new indices.

The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5071 amino acid residues, is encoded by a single gene, ryr-1/unc-68. The unc-68(kh30) mutation, isolated in an animal showing abnormal response to the anesthetic ketamine, has the substitution Ser1444Asn in CeRyR, predicted to be a phosphorylation site. To elucidate the function of the region of CeRyR, and to determine the localization of CeRyR in this animal, ten region-peptides were produced in Escherichia coli by using expression plasmids and eight antisera were raised against these fusion peptides. One antibody against the region corresponding to the kh30 mutation site enabled detection of CeRyR from mutant animals both in Western analysis and in situ. Specificity of this antiserum was demonstrated using Western analysis, which showed the full size and the partial size bands in wild-type and in the Tc1-induced deletion mutant animals, respectively, but no corresponding bands in unc-68 null mutant animals. CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining. CeRyR was found in the body wall, pharyngeal, vulval, anal and sex muscles of adult worms and also found to be present in embryonic muscle, but not in non-muscle cells. Two EF-hand motifs and the C terminus were demonstrated to be Ca2+ binding regions. On the basis of these results, we propose a model for the functional domains of CeRyR, which agrees well with the model of mammalian skeletal RyR, which is based on proteolysis and cross-linking analysis. We discuss the usefulness and limitations of the molecular dissection approach, which uses peptides and peptide-specific antibodies to determine the local structure and function of individual domains within a large molecule.

Genes participating in excitation-contraction coupling in Caenorhabditis elegans are cloned and these mutant phenotypes are characterized. These are the ryanodime receptor gene unc-68, troponin C gene pat-10 and tropomyosin gene lev-11. Unc-68 mutant animal is viable and has weak phenotype even in the null mutation. Contrary to our expectation, mutant analysis on pat-10 and lev-11 suggests that functional defects of the proteins affect not only function of muscle contraction but also muscle development of the animal. Experimental strategy and procedures are also useful for those who have interest in handling the model animal.

To overcome the limitations and misjudgments of conventional prediction of arrhythmic cardiotoxicity, we have developed an on-chip in vitro predictive cardiotoxicity assay using cardiomyocytes derived from human stem cells employing a constructive spatiotemporal two step measurement of fluctuation (short-term variability; STV) of cell's repolarization and cell-to-cell conduction time, representing two origins of lethal arrhythmia. Temporal STV of field potential duration (FPD) showed a potential to predict the risks of lethal arrhythmia originated from repolarization dispersion for false negative compounds, which was not correctly predicted by conventional measurements using animal cells, even for non-QT prolonging clinical positive compounds. Spatial STV of conduction time delay also unveiled the proarrhythmic risk of asynchronous propagation in cell networks, whose risk cannot be correctly predicted by single-cell-based measurements, indicating the importance of the spatiotemporal fluctuation viewpoint of in vitro cell networks for precise prediction of lethal arrhythmia reaching clinical assessment such as thorough QT assay.

An increasing body of evidence suggests that calmodulin (CaM) signaling is involved in the regulation of gene expression in cardiac hypertrophy (HT). According to recent report (Yamaguchi et al, 2007) mice expressing RyR2 mutant, that cannot bind CaM, develops cardiac HT. In an attempt to gain further insight into the role of RyR2-bound CaM in the process of development of HT, we have induced HT in neonatal rat cardiomyocytes by endothelin-1 (ET-1), and found distinct differences between the hypertrophied cells and control cells in several aspects. (1) The immuno-stain with anti-CaM antibody is chiefly localized in the cytoplasm of the control cells; whereas, in the HT cells, the stain is accumulated chiefly in the nucleus. (2) The cross linking/Western blot analysis shows that CaM is cross-linked with the RyR2 in control cells, but not in the HT cells. (3) We have prepared fluorescently labeled CaM by conjugating C34 mutant of CaM with Alexa 546, and introduced it into the myocytes using Bioporter. The exogenously introduced CaM-alexa is localized mainly in the cytoplasmic region in control cells, but within the nucleus in the HT cells. These findings suggest that stimulation of the neonatal cardiomyocytes with ET-1 dissociates RyR2-bound CaM from the receptor, then the dissociated CaM translocates to the nucleus to activate the HT gene program. In support of this conclusion, dantrolene (a specific blocker of RyR Ca2+ channel) inhibited HT in the ET-1-treated cells; at the same time prevented CaM translocation to the nucleus. Furthermore, an antibody raised against the CaM binding domain of RyR2, which was introduced into the cell via Bioporter, also prevented HT in the ET-1-treated cells. (Supported by HL072841)

Recent studies on cardiac hypertrophy animal model suggest that inter-domain interactions within the ryanodine receptor (RyR2) become defective concomitant with the development of hypertrophy (e.g. de-stabilization of the interaction between N-terminal and central domains of RyR2; T. Oda, M. Yano, T. Yamamoto, T. Tokuhisa, S. Okuda, M. Doi, T. Ohkusa, Y. Ikeda, S. Kobayashi, N. Ikemoto, M. Matsuzaki, Defective regulation of inter-domain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure, Circulation 111 (2005) 3400-3410). To determine if de-stabilization of the inter-domain interaction in fact causes hypertrophy, we introduced DPc10 (a peptide corresponding to the G(2460)-P(2495) region of RyR2, which is known to de-stabilize the N-terminal/central domain interaction) into rat neonatal cardiomyocytes by mediation of peptide carrier BioPORTER. After incubation for 24h the peptide induced hypertrophy, as evidenced by significant increase in cell size and [(3)H]leucine uptake. K201 or dantrolene, the reagents known to correct the de-stabilized inter-domain interaction to a normal mode, prevented the DPc10-induced hypertrophy. These results suggest that disruption of the normal N-terminal/central inter-domain interaction within the RyR2 is a causative mechanism of cardiomyocyte hypertrophy.

Ryanodine receptor (RyR) mutations linked with some congenital skeletal and cardiac diseases are localized to three easily definable regions: region 1 (N-terminal domain), region 2 (central domain), and a rather broad region 3 containing the channel pore. As shown in our recent studies, the interdomain interaction between regions 1 and 2 plays a critical role in channel regulation and pathogenesis. Here we present evidence that within region 3 there is a similar channel regulation mechanism mediated by an interdomain interaction. DP15, a peptide corresponding to RyR1 residues 4820-4841, produced significant activation of [ 3 H]ryanodine binding above threshold Ca 2+ concentrations (≥0.3 μM), but MH mutations (L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identify the DP15 binding site(s) within RyR 1 , DP15 (labeled with a fluorescent probe Alexa Fluor 680 and a photoaffinity cross-linker APG) was cross-linked to RyR 1 , and the site of cross-linking was identified by gel analysis of fluorescently labeled proteolytic fragments with the aid of Western blotting with site-specific antibodies. The shortest fluorescently labeled band was a 96 kDa fragment which was stained with an antibody directed to the region of residues 4114-4142 of RyR 1 , indicating that the interaction between the region of residues 4820-4841 adjacent to the channel pore and the 96 kDa segment containing the region of residues 4114-4142 is involved in the mechanism of Ca 2+ -dependent channel regulation. In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channel activation similar to that of DP15.

To explain the mechanism of pathogenesis of channel disorder in MH (malignant hyperthermia), we have proposed a model in which tight interactions between the N-terminal and central domains of RyR1 (ryanodine receptor 1) stabilize the closed state of the channel, but mutation in these domains weakens the interdomain interaction and destabilizes the channel. DP4 (domain peptide 4), a peptide corresponding to residues Leu2442–Pro2477 of the central domain, also weakens the domain interaction and produces MH-like channel destabilization, whereas an MH mutation (R2458C) in DP4 abolishes these effects. Thus DP4 and its mutants serve as excellent tools for structure–function studies. Other MH mutations have been reported in the literature involving three other amino acid residues in the DP4 region (Arg2452, Ile2453 and Arg2454). In the present paper we investigated the activity of several mutants of DP4 at these three residues. The ability to activate ryanodine binding or to effect Ca2+ release was severely diminished for each of the MH mutants. Other substitutions were less effective. Structural studies, using NMR analysis, revealed that the peptide has two α-helical regions. It is apparent that the MH mutations are clustered at the C-terminal end of the first helix. The data in the present paper indicates that mutation of residues in this region disrupts the interdomain interactions that stabilize the closed state of the channel.

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Understanding the importance of spatial and temporal regulation of cellular community size and shape are keys to resolve mechanisms in highly complex heart system by new quasi-in vivo assay, which can predict the phenomenon in vivo using in vitro assay. For that, an on-chip cell network cultivation system has been developed, in which field potential (FP) of cardiomyocyts in geometrically patterning chambers have been recorded with a multielectrode array (MEA) system.We have fabricated ring-shaped microelectrode having 1 or 2 mm in diameter and 50 μm in width, and cultivated cardiomyocytes from mouse embryonic hearts on the electrode with same closed circuit shape assisted by agarose microstructures. After the confirmation of regular propagation of beatings, we added a 1μM Astemizole which is well known to raise ventricular arrhythmias, and found that the profile of FP changed to the abnormal beating shape and then reached to the fibrillation shape [1]. To investigate the propagation pathway of excitation on abnormal beating, we constructed the circuit on 16 electrodes with area of 50x50 μm in a similar geometry to ring electrode assay. When abnormal beating happened on convoluted FPs obtained from all the 16 channels, one directional block on propagation were occurred and induced “re-entry” [2]. These results suggest that abnormal propagation, such as re-entry, causes abnormal fibrillation-like signal on the ring electrode.In conclusion, a simple quasi-in vivo ECG measurement assay has been proposed and developed and the results showed the typical arrhythmia profiles, in which both the temporal depolarization information and the spatial beating propagation information were appeared.[1] Nomura F, et al. J. Nanobiotechnology. 9, 39, 2011.[2] Kaneko T, et al. Jpn. J. Appl. Phys., 50, 070213, 2011.

We have evaluated the electrophysiological characteristics of a line-shaped network of a three-dimensionally controlled in vitro human cardiomyocyte assay (hCM line) against conventional cell clusters as the standard model (hCM cluster) from the viewpoint of quality control of sample variety and time–course stability. The beating intervals of the hCM line demonstrated a more stable uniformity of samples (846 ±130 ms, 15.3% fluctuation) and better time–course stability, whereas those of the hCM cluster showed a much larger variety of samples (2001 ±1127 ms, 56.3% fluctuation) and weaker time–course stability. The field potential amplitude of the hCM line also showed better uniformity of samples (629 ±428 µV, 68.0% fluctuation) against those of the hCM cluster (1984 ±2288 µV, 115.3% fluctuation). The results suggested the importance of the cell-network shape control for the uniformity and stability of the beating interval and the field potential amplitude. They also suggest that the hCM line can improve the reproducibility and accuracy of the samples, which is important for a functional human cardiotoxicity model.

To predict the risk of fatal arrhythmia induced by cardiotoxicity in the highly complex human heart system, we have developed a novel quasi-in vivo electrophysiological measurement assay, which combines a ring-shaped human cardiomyocyte network and a set of two electrodes that form a large single ring-shaped electrode for the direct measurement of irregular cell-to-cell conductance occurrence in a cardiomyocyte network, and a small rectangular microelectrode for forced pacing of cardiomyocyte beating and for acquiring the field potential waveforms of cardiomyocytes. The advantages of this assay are as follows. The electrophysiological signals of cardiomyocytes in the ring-shaped network are superimposed directly on a single loop-shaped electrode, in which the information of asynchronous behavior of cell-to-cell conductance are included, without requiring a set of huge numbers of microelectrode arrays, a set of fast data conversion circuits, or a complex analysis in a computer. Another advantage is that the small rectangular electrode can control the position and timing of forced beating in a ring-shaped human induced pluripotent stem cell (hiPS)-derived cardiomyocyte network and can also acquire the field potentials of cardiomyocytes. First, we constructed the human iPS-derived cardiomyocyte ring-shaped network on the set of two electrodes, and acquired the field potential signals of particular cardiomyocytes in the ring-shaped cardiomyocyte network during simultaneous acquisition of the superimposed signals of whole-cardiomyocyte networks representing cell-to-cell conduction. Using the small rectangular electrode, we have also evaluated the response of the cell network to electrical stimulation. The mean and SD of the minimum stimulation voltage required for pacing (V Min) at the small rectangular electrode was 166±74 mV, which is the same as the magnitude of amplitude for the pacing using the ring-shaped electrode (179±33 mV). The results showed that the addition of a small rectangular electrode into the ring-shaped electrode was effective for the simultaneous measurement of whole-cell-network signals and single-cell/small-cluster signals on a local site in the cell network, and for the pacing by electrical stimulation of cardiomyocyte networks.

Quantitative evaluation of mechanophysiological responses of cardiomyocytes has become more important for more precise prediction of cardiotoxicity. For the accurate detection of cardiomyocyte contraction, we have developed an on-chip single-cell-shape control technology on the basis of an agarose microchamber system and an on-chip optical image analysis system that records the contractile motions of cardiomyocytes with noninvasive/nondestructive measurement for long-term experiments. Using this on-chip single-cell-shape control technology, the shape of single cardiomyocytes was controlled by seeding the cells in 21-µm-radius (circular) or 20×70 µm2 (rectangular) agarose microchambers. To detect the contractility of cardiomyocytes, the cells were labeled with microbeads attached onto the surface of target cells and the motion of beads was acquired and analyzed using a newly developed wider-depth-of-field optics equipped with a 1/100 s high-speed digital camera. Mechanophysiological properties such as displacement and direction of movement were obtained using a real-time processing system module at spatial and temporal resolutions of 0.15 µm and 10 ms, respectively. Comparisons of displacement and direction of contraction between circular and rectangular cardiomyocytes indicated that the rectangular cardiomyocytes tended to contract along the longitudinal direction as in a real heart. This result suggests that the shape of cells affected the function of cells. The on-chip single-cell-shape control technology and optical image analysis system enable the detection of the motion of contraction of single-shape-controlled cardiomyocytes, and are expected to be applicable to the more precise prediction of cardiotoxicity.

Re-entry of excitation in the heart is one of the abnormal phenomena to cause the lethal arrhythmia such as torsade de pointes (TdP) and is thought to be induced by the looped structure of excitation conduction pathway. For adaptable in vitro preclinical strategies to evaluate global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal arrhythmia measurement using circuit-shaped cardiomyocyte network model has been developed. The electrocardiogram (ECG)-like field potential data, which includes the information both of the repolarization and of the conductance abnormality, was acquired from the self-convoluted extracellular field potential (FP) profiles of a lined-up cardiomyocyte network on a ring-shaped electrode in an agarose microchamber. When E-4031 (hERG blocker) or Astemizole (TdP positive but false-negative drug traditional in vitro assay) were applied to the closed-loop cardiomyocyte network, self-convoluted FP profile of normal beating changed into an abnormal waveform, including early afterdepolarization (EAD) like or TdP-like abnormal waveform at 1 μM in either case (2 out of 8 samples in E-4031 and 8 out of 10 samples in Astemizole), and especially followed by fibrillation-like arrhythmia and arrest in Astemizole. Self-convoluted FP duration prolongation and its fluctuation increase were also observed according to the increase of E-4031 concentration (188±21% prolongation of FPD corrected with beating rate and 168±42% increase of STV at 1 μM, mean±SE, n=6). The results indicate that the self-convoluted FPs of the ring-shaped cardiomyocyte network assay could be used for quasi-ECG measurement to estimate QT prolongation and arrhythmic potential including the effects on both repolarization process and the conductance abnormality of cardiomyocyte.

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.

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.

Special attention is directed to the potential application of human induced pluripotent stem (iPS) cell-derived cardiomyocytes for cardiac safety pharmacology and toxicology with fewer legal and ethical issues. Supply of commercially available products enables many researchers to test the utility in their own systems. Although variations of electrophysiological properties have been reported among pluripotent cell lines by classifying cardiac subtypes into nodal, atrial and ventricular cells, most cells are spontaneously contracting, which makes difficult to be implicated in adult human hearts. Thus, we sought objective description on action potential (AP) parameters recorded from perforated patch-clamped iCell cardiomyocytes (iCell-CMs, AJ/CDI) by systematically comparing with those from human ES-derived cardiomyocytes (hES-CMs, Cellartis). Mean +/- SE values of APD50 in iCell-CMs (382 +/- 38 ms, n=36) were significantly longer than those in hES-CMs (278 +/- 28 ms n=64), while APD90 in iCell-CMs (1210 +/- 346 ms, n=36) tended to be longer than those in hES-CMs (776 +/- 136 ms n=64). As for APD90, despite of the large sample size, there was no statistical significance between iPS-CM and hES-CMs. Detailed analysis of the variation by Gaussian fitting revealed that marked differences in shapes of probability distribution between iPS-CMs and hES-CMs. Although further studies are necessary to know if the variation of AP parameters affect drug responses, our data provide important information for cardiac safety assessments.