On-chip spatiotemporal electrophysiological analysis of human stem cell derived cardiomyocytes enables quantitative assessment of proarrhythmia in drug development.

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.

A distribution analysis of action potential parameters obtained from patch-clamped human stem cell-derived cardiomyocytes.

We investigated electrophysiological properties of human induced-pluripotent-stem-cell-derived and embryonic-stem-cell-derived cardiomyocytes, and analyzed action potential parameters by plotting their frequency distributions. In the both cell lines, the distribution analysis revealed that histograms of maximum upstroke velocity showed two subpopulations with similar intersection values. Sub-populations with faster maximum upstroke velocity showed significant prolongation of action potential durations by application of E-4031, whereas others did not, which may be partly due to shallower maximum diastolic potentials. We described electrophysiological and pharmacological properties of stem-cell-derived cardiomyocytes in the respective sub-populations, which provides a way to characterize diverse electrical properties of stem-cell-derived cardiomyocytes systematically.

On-chip in vitro cell-network pre-clinical cardiac toxicity using spatiotemporal human cardiomyocyte measurement on a chip.

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.

Defective regulation of the ryanodine receptor induces hypertrophy in cardiomyocytes.

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.

The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

Peptide probe study of the role of interaction between the cytoplasmic and transmembrane domains of the ryanodine receptor in the channel regulation mechanism.

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 [3H]ryanodine binding above threshold Ca2+ concentrations (>or=0.3 microM), but MH mutations (L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identify the DP15 binding site(s) within RyR1, DP15 (labeled with a fluorescent probe Alexa Fluor 680 and a photoaffinity cross-linker APG) was cross-linked to RyR1, 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 RyR1, 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 Ca2+-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.

Malignant hyperthermia mutation sites in the Leu2442-Pro2477 (DP4) region of RyR1 (ryanodine receptor 1) are clustered in a structurally and functionally definable area.

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 a-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.

Dantrolene stabilizes domain interactions within the ryanodine receptor.

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.

Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans.

The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5,071 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 Ca(2+) 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.