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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.

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)

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.

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.