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Parameter optimization (PO) methods to determine the ionic current composition of experimental cardiac action potential (AP) waveform have been developed using a computer model of cardiac membrane excitation. However, it was suggested that fitting a single AP record in the PO method was not always successful in providing a unique answer because of a shortage of information. We found that the PO method worked perfectly if the PO method was applied to a pair of a control AP and a model output AP in which a single ionic current out of six current species, such as I Kr , I CaL , I Na , I Ks , I Kur or I bNSC was partially blocked in silico. When the target was replaced by a pair of experimental control and I Kr -blocked records of APs generated spontaneously in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the simultaneous fitting of the two waveforms by the PO method was hampered to some extent by the irregular slow fluctuations in the V m recording and/or sporadic alteration in AP configurations in the hiPSC-CMs. This technical problem was largely removed by selecting stable segments of the records for the PO method. Moreover, the PO method was made fail-proof by running iteratively in identifying the optimized parameter set to reconstruct both the control and the I Kr -blocked AP waveforms. In the lead potential analysis, the quantitative ionic mechanisms deduced from the optimized parameter set were totally consistent with the qualitative view of ionic mechanisms of AP so far described in physiological literature., (© 2024. The Author(s).)

In this article, we review studies which have been conducted to investigate the hormonal influence on metamorphosis in bullfrog (Rana catesbeiana) and Japanese toad (Bufo japonicus) larvae, in addition to studies conducted on the hormonal and pheromonal control of reproductive behavior in red-bellied newts (Cynops pyrrhogaster). Metamorphosis was studied with an emphasis on the roles of prolactin (PRL) and thyrotropin (TSH). The release of PRL was shown to be regulated by thyrotropin-releasing hormone (TRH) and that of TSH was evidenced to be regulated by corticotropin-releasing factor. The significance of the fact that the neuropeptide that controls the secretion of TSH is different from those encountered in mammals is discussed in consideration of the observation that the release of TRH, which stimulates the release of PRL, is enhanced when the animals are subjected to a cold temperature. Findings that were made by using melanin-rich cells of Bufo embryos and larvae, such as the determination of the origin of the adenohypophyseal primordium, identification of the pancreatic chitinase, and involvement of the rostral preoptic recess organ as the hypothalamic inhibitory center of α-melanocyte-stimulating hormone (α-MSH) secretion, are mentioned in this article. In addition, the involvement of hormones in eliciting courtship behavior in male red-bellied newts and the discovery of the peptide sex pheromones and hormonal control of their secretion are also discussed in the present article., (© 2023 Japanese Society of Developmental Biologists.)

Background: CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca 2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays., Methods: We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca 2+ affinities of CaM using recombinant proteins., Results: We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca 2+ waves than the other lines in association with increased Ca 2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [ 3 H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca 2+ ] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca 2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca 2+ waves in E46K-cardiomyocytes., Conclusions: We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.

Background: A missense mutation in the α1c subunit of voltage-gated L-type Ca 2+ channel-coding CACNA1C-E1115K, located in the Ca 2+ selectivity site, causes a variety of arrhythmogenic phenotypes., Objective: We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs)., Methods: We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9-mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model., Results: Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca 2+ current density and impaired Ca 2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na + channel current (I NaL ) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of I NaL . An in silico study recapitulated the in vitro electrophysiological properties., Conclusion: Our iPSC-based analysis in CACNA1C-E1115K with disrupted Ca V 1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous I NaL , thereby synergistically contributing to the arrhythmogenic phenotype., (Copyright © 2022 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Urodele amphibians have the ability to regenerate several organs, including the brain. For this reason, the research on neurogenesis in these species after ablation of some parts of the brain has markedly progressed. However, detailed information on the characteristics and fate of proliferated cells as well as the function of newly generated neurons under normal conditions is still limited. In this study, we focused on investigating the proliferative and neurogenic zones as well as the fate of proliferated cells in the adult brain of the Japanese red-bellied newt to clarify the significance of neurogenesis in adulthood. We found that the proximal region of the lateral ventricles in the telencephalon and the preoptic area in the diencephalon were the main sites for continuous cell proliferation in the adult brain. Furthermore, we characterized proliferative cells and analyzed neurogenesis through a combination of 5-ethynyl-2'-deoxyuridine (EdU) labeling and immunohistochemistry using antibodies against the stem cell marker Sox2 and neuronal marker NeuN. Twenty-four hours after EdU injection, most of the EdU-positive cells were Sox2-immunopositive, whereas, EdU-positive signals and NeuN-immunoreactivities were not colocalized. Two months after EdU injection, the colocalization ratio of EdU-positive signals with Sox2-immunoreactivities decreased to approximately 10%, whereas the ratio of colocalization of EdU-positive signals with NeuN-immunoreactivities increased to approximately 60%. Furthermore, a portion of the EdU-incorporated cells developed into γ-aminobutyric acid-producing cells, which are assumed to function as interneurons. On the basis of these results, the significance of newly generated neurons was discussed with special reference to their reproductive behavior., (© 2022 Japanese Society of Developmental Biologists.)

Premature cardiac myocytes derived from human induced pluripotent stem cells (hiPSC-CMs) show heterogeneous action potentials (APs), probably due to different expression patterns of membrane ionic currents. We developed a method for determining expression patterns of functional channels in terms of whole-cell ionic conductance (G x ) using individual spontaneous AP configurations. It has been suggested that apparently identical AP configurations can be obtained using different sets of ionic currents in mathematical models of cardiac membrane excitation. If so, the inverse problem of G x estimation might not be solved. We computationally tested the feasibility of the gradient-based optimization method. For a realistic examination, conventional 'cell-specific models' were prepared by superimposing the model output of AP on each experimental AP recorded by conventional manual adjustment of G x s of the baseline model. G x s of 4-6 major ionic currents of the 'cell-specific models' were randomized within a range of ± 5-15% and used as an initial parameter set for the gradient-based automatic G x s recovery by decreasing the mean square error (MSE) between the target and model output. Plotting all data points of the MSE-G x relationship during optimization revealed progressive convergence of the randomized population of G x s to the original value of the cell-specific model with decreasing MSE. The absence of any other local minimum in the global search space was confirmed by mapping the MSE by randomizing G x s over a range of 0.1-10 times the control. No additional local minimum MSE was obvious in the whole parameter space, in addition to the global minimum of MSE at the default model parameter., (© 2022. The Author(s).)

Timothy syndrome (TS) is a rare pleiotropic disorder associated with long QT syndrome, syndactyly, dysmorphic features, and neurological symptoms. Several variants in exon 8 or 8a of CACNA1C, a gene encoding the α-subunit of voltage-gated Ca 2+ channels (Ca v 1.2), are known to cause classical TS. We identified a p.R412M (exon 9) variant in an atypical TS case. The aim of this study was to examine the functional effects of CACNA1C p.R412M on Ca V 1.2 in comparison with those of p.G406R. The index patient was a 2-month-old female infant who suffered from a cardio-pulmonary arrest in association with prolonged QT intervals. She showed dysmorphic facial features and developmental delay, but not syndactyly. Interestingly, she also presented recurrent seizures from 4 months. Genetic tests identified a novel heterozygous CACNA1C variant, p.R412M. Using heterologous expression system with HEK-293 cells, analyses with whole-cell patch-clamp technique revealed that p.R412M caused late Ca 2+ currents by significantly delaying Ca V 1.2 channel inactivation, consistent with the underlying mechanisms of classical TS. A novel CACNA1C variant, p.R412M, was found to be associated with atypical TS through the same mechanism as p.G406R, the variant responsible for classical TS., (© 2022. The Author(s).)

Pregnancy causes changes in the uterus, such as increased cell volume and altered water content. However, the mechanisms that protect the structure and maintain the function of uterine smooth muscle cells against these changes during pregnancy have not been clarified. This study focused on the volume-regulated anion channel (VRAC), which opens with cell swelling under low osmotic pressure and releases Cl - ions and various organic osmolytes to resist cell swelling and regulates a wide range of biological processes such as cell death. In this study, myometrial smooth muscle (MSM) tissues and cells (MSMCs) were collected from non-pregnant and pregnant mice. Using western blotting and immunocytochemistry, leucine-rich repeat containing protein 8A (LRRC8A), an essential membrane protein that constitutes part of the VRAC, was determined to be diffused throughout MSMCs including in the cell membrane. Patch-clamp experiments were performed to investigate the electrophysiology of swelling-induced Cl - currents (I Cl, swell ) mediated by the VRAC. No significant changes between non-pregnancy and pregnancy groups were observed in either the expression density of LRRC8A or the current density of I Cl, swell , however the presence of LRRC8A on the cell membrane was significantly increased in the third trimester of pregnancy compared to the non-pregnancy. This study suggests that the VRAC may play a role, such as maintaining cellular homeostasis in the pregnant MSM.

Propofol is a broadly used intravenous anesthetic agent that can cause cardiovascular effects, including bradycardia and asystole. A possible mechanism for these effects is slowing cardiac pacemaker activity due to inhibition of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. However, it remains unclear how propofol affects the allosteric nature of the voltage- and cAMP-dependent gating mechanism in HCN channels. To address this aim, we investigated the effect of propofol on HCN channels (HCN4 and HCN2) in heterologous expression systems using a whole-cell patch clamp technique. The extracellular application of propofol substantially suppressed the maximum current at clinical concentrations. This was accompanied by a hyperpolarizing shift in the voltage dependence of channel opening. These effects were significantly attenuated by intracellular loading of cAMP, even after considering the current modification by cAMP in opposite directions. The differential degree of propofol effects in the presence and absence of cAMP was rationalized by an allosteric gating model for HCN channels, where we assumed that propofol affects allosteric couplings between the pore, voltage-sensor, and cyclic nucleotide-binding domain (CNBD). The model predicted that propofol enhanced autoinhibition of pore opening by unliganded CNBD, which was relieved by the activation of CNBD by cAMP. Taken together, these findings reveal that propofol acts as an allosteric modulator of cAMP-dependent gating in HCN channels, which may help us to better understand the clinical action of this anesthetic drug.

Delayed rectifier K + current (I Ks ) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of I Ks in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and I Ks current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of I Ks was examined using immunocytochemistry and Western blotting. KCNQ1, a I Ks pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The I Ks current in PVC was markedly enhanced by both β 1 - and β 2 -adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β 2 -adrenoceptor stimulation than β 1 -adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in I Ks were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the I Ks current was increased by α 1 -adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective I Ks blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α 1 -adrenoceptor on I Ks in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of I Ks might increase during sympathetic excitation under in vivo conditions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gastric carcinoma with lymphoid stroma (GCLS), a rare subset of gastric cancer, has a low frequency of lymphovascular invasion and a relatively better prognosis compared with conventional gastric cancer. We herein report a rare case of early GCLS successfully treated by endoscopic submucosal dissection alone. The lesion was located in the upper gastric body and approximately 9 mm in size. We assessed that the lesion was within an absolute indication for endoscopic resection. We performed endoscopic submucosal dissection and succeeded in en bloc resection. A histopathological assessment disclosed that the carcinoma was poorly differentiated with massive infiltration of lymphocyte and invaded the submucosal layer massively at 1000 μm in depth. There were no visible lymphovascular invasions in the specimen. Since the Epstein-Barr virus (EBV)-encoded small RNA in situ hybridization revealed that cancer cells were positive for EBV, the patient was finally diagnosed with EBV-positive GCLS. We persuaded the patient to receive an additional surgery; however, the patient refused to undergo it. The patient has been followed for more than 5 years without recurrence., (© 2021. Japanese Society of Gastroenterology.)

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na + current and Na v 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na + current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Na v 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Na v 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS., (© 2020 Federation of American Societies for Experimental Biology.)

Objective: The Spontaneously Diabetic Torii (SDT) fatty rat, established by introducing the fa allele (obesity gene) of the Zucker fatty rat into the SDT rat genome, is a new model of obese type 2 diabetes. We studied the pathologic features of diabetic retinopathy (DR) in this animal., Methods: The eyes of SDT fatty, SDT (controls), and Sprague Dawley (SD) rats (normal controls) were enucleated at 8, 16, 24, 32, and 40 weeks of age ( n = 5-6 for each rat type at each age). The retinal thicknesses, numbers of retinal folds, and choroidal thicknesses were evaluated. Immunostaining for glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) was performed. Quantitative analyses of the immunopositive regions were performed using a cell-counting algorithm., Results: The retinas tended to be thicker in the SDT fatty rats and SDT rats than in the SD rats; the choroids tended to be thicker in the SDT fatty rats than in the SD rats. The retinal folds in the SDT fatty rats developed earlier and were more severe than in the SDT rats. Quantitative analyses showed that the GFAP- and VEGF-positive regions in the retinas of the SDT fatty rats were significantly larger than those of the SDT rats., Conclusions: SDT fatty rats developed more severe DR earlier than the SDT rats. The SDT fatty rats might be useful as a type 2 diabetes animal model to study DR., Competing Interests: All authors declare that there are no conflicts of interest regarding the publication of this paper. Drs. Ohta and Sasase are employees of Japan Tobacco Inc., (Copyright © 2019 Yoshiaki Tanaka et al.)

Background: KCNQ1-T587M is a C-terminal mutation correlated with severe phenotypes of long QT syndrome (LQTS). However, functional analysis of KCNQ1 channels with the T587M mutation showed a mild genotype in the form of haploinsufficiency in a heterologous expression system. This study sought to explore the molecular mechanism underlying the phenotype-genotype dissociation of LQTS patients carrying the KCNQ1-T587M mutation., Methods: cDNAs for wild-type (WT) and KCNQ1 mutations (R259C and T587M) were transiently transfected into HEK293 cells stably expressing hERG (hERG-HEK), and whole-cell patch-clamp technique was performed to examine the effect of KCNQ1 mutations on I Kr -like currents. In addition, fluorescence resonance energy transfer (FRET) was conducted to demonstrate the molecular interaction between KCNQ1 and hERG when co-expressed in HEK293 cells., Results: KCNQ1-T587M mutation produced a significant (p<0.01) decrease in I Kr -like tail current densities without affecting the gating kinetics, while KCNQ1-R259C mutation had no significant effect on the I Kr -like tail current densities. Consistent with this result, FRET experiments demonstrated that both KCNQ1-WT and -R259C interacted with hERG in the cytosol and on the plasma membrane; however, the interaction between KCNQ1-T587M and hERG was observed only in the cytosol, and hERG proteins were seldom transported to the cell membrane, suggesting that the KCNQ1-T587M mutation impaired the trafficking of hERG to the cell membrane., Conclusions: The disruption of hERG trafficking caused by the KCNQ1-T587M mutation is likely the reason why some patients exhibit severe LQTS phenotypes., (Copyright © 2018 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E 2 (PGE 2 ) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE 2 release and was attenuated by inhibition of either phospholipase A 2 or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.

We herein report a case of a 31-year-old Japanese man who simultaneously had a positive influenza A virus antigen test result and Vogt-Koyanagi-Harada disease (VKHD), demonstrated by both diffuse multiple early hyperfluorescent points on fluorescein fundus photography and serous retinal detachments on optical coherence tomography. He had meningitis. It was difficult to determine whether the main cause of meningitis was influenza A or VKHD. After initial treatment with peramivir for influenza A and then methylprednisolone pulse with subsequent corticosteroid therapy for VKHD, his symptoms improved gradually. These findings suggest that influenza A virus infection contributes to the onset or exacerbation of VKHD.

Dr. Wei-Guang Ding's given name and family name were inadvertently interchanged initially. The correct names are as shown above.

The sustained inward Na + current (I st ) identified in the sinoatrial node (SAN) cell has been suggested to play a pivotal role in cardiac pacemaking. However, the composition of cells in the SAN is heterogeneous and cell-to-cell variability in the magnitude of I st remains to be fully characterized. The present study investigated the current density of I st in morphologically different types of pacemaker cells dissociated from guinea pig SAN. I st was preferentially detected in spontaneously active spindle or spider-shaped cells, but was less well expressed in larger-sized elongated spindle-type cells and practically absent in clearly striated atrial-like cells, despite clear expression of the funny current (I f ). The current density of I st in spindle and spider cells varied from 0.7 to 1.6 pA pF -1 and was significantly reduced in non-beating cells with similar morphologies. By linear regression analysis, we identified a positive correlation between the current densities of I st and the L-type Ca 2+ current (I Ca,L ), which was specifically observed in spindle and spider cells. These cells exhibited a more negative voltage for half maximal I Ca,L activation than atrial-like cells, suggesting a variable ratio between Ca V 1.2- and Ca V 1.3-mediated I Ca,L in SAN cells. Consistent single-cell transcript measurements confirmed a higher relative expression of Ca V 1.3, which activates at more negative potentials, in spindle cells than in atrial-like cells. Taken together, these results can be interpreted as indicating that I st plays a specific role in primary pacemaker cells and that its presence is closely correlated with functional levels of Ca V 1.3-mediated I Ca,L .

Congenital long QT syndrome (LQTS) caused by compound mutations is usually associated with more severe clinical phenotypes. We identified a LQTS family harboring three compound mutations in different genes (KCNQ1-R174C, hERG-E1039X and SCN5A-E428K). KCNQ1-R174C, hERG-E1039X and SCN5A-E428K mutations and/or relevant wild-type (WT) cDNAs were respectively expressed in mammalian cells. I Ks -like, I Kr -like, I Na -like currents and the functional interaction between KCNQ1-R174C and hERG-E1039X channels were studied using patch-clamp and immunocytochemistry techniques. (1) Expression of KCNQ1-R174C alone showed no I Ks . Co-expression of KCNQ1-WT + KCNQ1-R174C caused a loss-of-function in I Ks and blunted the activation of I Ks in response to isoproterenol. (2) Expression of hERG-E1039X alone and co-expression of hERG-WT + hERG-E1039X negatively shifted inactivation curves and decelerated the recovery time from inactivation. (3) Expression of SCN5A-E428K increased peak I Na , but had no effect on late I Na . (4) I Ks and I Kr interact, and hERG-E1039X caused a loss-of-function in I Ks . (5) Immunocytochemical studies indicated that KCNQ1-R174C is trafficking defective and hERG-E1039X is defective in biosynthesis/degradation, but the abnormities were rescued by co-expression with WT. Thus, KCNQ1-R174C and hERG-E1039X disrupted I Ks and I Kr functions, respectively. The synergistic lesion, caused by KCNQ1-R174C and hERG-E1039X in I Ks , is very likely why patients showed more severe phenotypes in the compound mutation case.

Background: TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, Na V 1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated with reduced Na V 1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of Na V 1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of Na V 1.5., Conclusions: This study successfully demonstrated diminished sodium currents resulting from lower Na V 1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.

The spontaneous activity of sinoatrial node (SAN) pacemaker cells is generated by a functional interplay between the activity of ionic currents of the plasma membrane and intracellular Ca 2+ dynamics. The molecular correlate of a dihydropyridine (DHP)-sensitive sustained inward Na + current (I st ), a key player in SAN automaticity, is still unknown. Here we show that I st and the L-type Ca 2+ current (I Ca,L ) share Ca V 1.3 as a common molecular determinant. Patch-clamp recordings of mouse SAN cells showed that I st is activated in the diastolic depolarization range, and displays Na + permeability and minimal inactivation and sensitivity to I Ca,L activators and blockers. Both Ca V 1.3-mediated I Ca,L and I st were abolished in Ca V 1.3-deficient (Ca V 1.3 -/- ) SAN cells but the Ca V 1.2-mediated I Ca,L current component was preserved. In SAN cells isolated from mice expressing DHP-insensitive Ca V 1.2 channels (Ca V 1.2 DHP-/- ), I st and Ca V 1.3-mediated I Ca,L displayed overlapping sensitivity and concentration-response relationships to the DHP blocker nifedipine. Consistent with the hypothesis that Ca V 1.3 rather than Ca V 1.2 underlies I st , a considerable fraction of I Ca,L was resistant to nifedipine inhibition in Ca V 1.2 DHP-/- SAN cells. These findings identify Ca V 1.3 channels as essential molecular components of the voltage-dependent, DHP-sensitive I st Na + current in the SAN.

The male red-bellied newt (Cynops pyrrhogaster) approaches the female's cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.

Human Kv1.5 channels (hKv1.5) conduct the ultra-rapid delayed rectifier potassium current (I Kur ), which plays an important role in action potential repolarization of atrial myocytes. The present study was undertaken to examine the effects of acidic pH on hKv1.5 wild-type (WT) and its pore mutant channels heterologously expressed in Chinese hamster ovary (CHO) cells using site-directed mutagenesis combined with whole-cell patch-clamp technique. Both extracellular and intracellular acidifications equally and reversely reduced the amplitude of hKv1.5 currents. The extracellular acidification significantly shifted the voltage dependence of current activation to more depolarized potentials and accelerated deactivation kinetics of the current. The ancillary β subunits Kvβ1.3 and Kvβ1.2, known to modify the pharmacological sensitivities of hKv1.5, enhanced the extracellular proton-induced inhibitory effect on hKv1.5 current. In addition, several mutants (T462C, T479A, T480A, and I508A) exhibited significantly higher sensitivity to acidic pH-induced inhibition compared with WT channel, whereas the inhibitory effect of acidic pH was markedly reduced in H463G mutant. These observations indicate that (1) extracellular acidification modifies hKv1.5 gating and activity, (2) β subunits and several residues (T462, T479, T480, and I508) play critical roles in determining the sensitivity of the channel to acidic exposure, and (3) H463 may be a critical sensor for the channel inhibition by extracellular protons.

Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine-induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3-30 mM) induced spherical protrusions on the cell surface (so called "membrane blebbing") in a time- and concentration-dependent manner. The concentration-response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non-ionized lidocaine being involved in the bleb formation. ROCK (Rho-kinase) inhibitors Y-27632 and fasudil completely prevented the lidocaine-induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase-3 levels were unchanged by 10 mM lidocaine (p = 0.325) and a caspase inhibitor z-VAD-fmk did not affect the lidocaine-induced blebbing (p = 0.964). GTP-RhoA levels were significantly increased (p < 0.001), but Rho inhibitor-1 failed to suppress the membrane blebbing (p = 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p < 0.001) and in situ chondrocytes (p < 0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin-II inhibitor blebbistatin (p < 0.001). These findings suggest that lidocaine induces ROCK-dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:754-762, 2016., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose: To present the characteristics and pathology of a patient with congenital achromatopsia., Patient and Methods: The patient was a 22-year-old Japanese woman who was 8 years old when she first visited our clinic. Comprehensive ophthalmic examinations including visual acuity measurements, perimetry, optical coherence tomography (OCT), fundus autofluorescence (FAF) imaging, electroretinography (ERG), and color vision tests were performed. Her genomic DNA was used as the template for the amplification of exons of five candidate genes for achromatopsia; CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H, and the amplified products were sequenced. A missense mutation, found in the CNGA3, was studied both electrophysiologically and biochemically., Results: Her phenotype was typical of congenital complete achromatopsia. She was followed for 14 years, and her vision and fundus findings were stable. However, the scotopic ERG b-waves at age 22 were smaller than those at age 8, and her FAF images showed increased autofluorescence in both maculae. Genetic examinations revealed combined heterozygous mutations of c.997&#95;998delGA and p.M424V in the CNGA3 gene. The homomeric channel consisting of the CNGA3 subunit with the p.M424V mutation had a weak cGMP-activated current in patch-clamp recordings. In heterologous expression analyses, the expression at the cell surface of the mutant CNGA3 subunit was about 28 % of the wild type., Conclusions: The two novel mutations found in the CNGA3 gene, c.997&#95;998delGA and p.M424V, can cause complete achromatopsia. The vision of the patient was stationary until the third decade of life although the FAF was altered at the age of 22 years.

We evaluated the features of diabetic retinal and choroidal edema in Spontaneously Diabetic Torii (SDT) rats. We measured the retinal and choroidal thicknesses in normal Sprague-Dawley (SD) rats (n = 9) and SDT rats (n = 8). The eyes were enucleated 40 weeks later after they were diagnosed with diabetes, and 4-micron sections were cut for conventional histopathologic studies. The mean retinal and choroidal thicknesses were significantly thicker in the SDT rats than in the normal SD rats. The choroidal thickness was correlated strongly with the retinal thickness in both rat models. Diabetic retinopathy (DR) and diabetic choroidopathy appeared as edema in the SDT rats. The retinal thickness was correlated strongly with the choroidal thickness in the SDT rats, which is an ideal animal model of both DR and choroidopathy.

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7α-hydroxypregnenolone (7α-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7α-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7α-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Simultaneous pulmonary metastases from different primary tumors to the same lobe are extremely rare, and we herein report the case. Surgical specimen of the pulmonary metastasis from colon cancer contained two additional nodules that were confirmed as metastases from prostate cancer. Pulmonary metastasis from prostate cancer rarely forms nodules, and there is a discrepancy in the incidence of pulmonary metastases between autopsy and clinical findings. This case suggests that different malignant tumors could simultaneously metastasize to the same pulmonary lobe, and more pulmonary metastases from prostate cancer might exist than expected.

The human ether-a-go-go-related gene (HERG) potassium current (IHERG) has been shown to decrease in amplitude following stimulation with Gq protein-coupled receptors (GqRs), such as α1-adrenergic and M1-muscarinic receptors (α1R and M1R, respectively), at least partly via the reduction of membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The present study was designed to investigate the modulation of HERG channels by PI(4,5)P2 and phosphatidylinositol4-phosphate 5-kinase (PI(4)P5-K), a synthetic enzyme of PI(4,5)P2. Whole-cell patch-clamp recordings were used to examine the activity of HERG channels expressed heterologously in Chinese Hamster Ovary cells. The stimulation of α1R with phenylephrine or M1R with acetylcholine decreased the amplitude of IHERG accompanied by a significant acceleration of deactivation kinetics and the effects on IHERG were significantly attenuated in cells expressing PI(4)P5-K. The density of IHERG in cells expressing GqRs alone was significantly increased by the coexpression of PI(4)P5-K without significant differences in the voltage dependence of activation and deactivation kinetics. The kinase-deficient substitution mutant, PI(4)P5-K-K138A did not have these counteracting effects on the change in IHERG by M1R stimulation. These results suggest that the current density of IHERG is closely dependent on the membrane PI(4,5)P2 level, which is regulated by PI(4)P5-K and GqRs and that replenishing PI(4,5)P2 by PI(4)P5-K recovers IHERG., (Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.)

A 51-year-old Japanese woman developed candidemia as an outpatient secondary to a Candida albicans upper urinary tract infection complicated by previously undiagnosed type 2 diabetes mellitus with poor glycemic control and ureterolithiasis. The patient did not have any risk factors typically associated with candidemia, such as an indwelling vascular catheter, parenteral nutrition or broad-spectrum antibiotic use. During the clinical course, her condition was complicated by unilateral candida endophthalmitis, which progressed despite the administration of systemic antifungal agents and ultimately required vitreous surgery. The etiology of candidemia in this patient and the reason she developed progressive ocular symptoms after starting antifungal treatment are reviewed.

Aims: CACNA1C mutations have been reported to cause LQTS type 8 (LQT8; Timothy syndrome), which exhibits severe phenotypes, although the frequency of patients with LQT8 exhibiting only QT prolongation is unknown. This study aimed to elucidate the frequency of CACNA1C mutations in patients with long QT syndrome (LQTS), except those with Timothy syndrome and investigate phenotypic variants., Methods and Results: CACNA1C gene screening was performed in 278 probands negative for LQTS-related gene mutations. Functional analysis of mutant channels using a whole-cell patch-clamp technique was also performed. Using genetic screening, we identified five novel CACNA1C mutations: P381S, M456I, A582D, R858H, and G1783C in seven (2.5%) unrelated probands. Seven mutation carriers showed alternative clinical phenotypes. Biophysical assay of CACNA1C mutations revealed that the peak calcium currents were significantly larger in R858H mutant channels than those of wild-type (WT). In contrast, A582D mutant channels displayed significantly slower inactivation compared with WT. The two mutant channels exerted different gain-of-function effects on calcium currents., Conclusion: In patients with LQTS, the frequency of CACNA1C mutations was higher than reported. Even without typical phenotypes of Timothy syndrome, CACNA1C mutations may cause QT prolongation and/or fatal arrhythmia attacks., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system., (© 2014 Wiley Periodicals, Inc.)