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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jphs.2024.07.007

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DOI： 10.1016/j.jphs.2024.05.002

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Language：English   Publisher：Journal of Pharmacological Sciences  

Sulfur-based redox signaling has long attracted attention as critical mechanisms underlying the development of cardiac diseases and resultant heart failure. Especially, post-translational modifications of cysteine (Cys) thiols in proteins mediate oxidative stress-dependent cardiac remodeling including myocardial hypertrophy, senescence, and interstitial fibrosis. However, we recently revealed the existence of Cys persulfides and Cys polysulfides in cells and tissues, which show higher redox activities than Cys and substantially contribute to redox signaling and energy metabolism. We have established simple evaluation methods that can detect polysulfides in proteins and inorganic polysulfides in cells and revealed that polysulfides abundantly expressed in normal hearts are dramatically catabolized by exposure to ischemic/hypoxic and environmental electrophilic stress, which causes vulnerability of the heart to mechanical load. Accumulation of hydrogen sulfide, a nucleophilic catabolite of persulfides/polysulfides, may lead to reductive stress in ischemic hearts, and perturbation of polysulfide catabolism can improve chronic heart failure after myocardial infarction in mice. This review focuses on the (patho)physiological role of sulfur metabolism in hearts, and proposes that sulfur catabolism during ischemic/hypoxic stress has great potential as a new therapeutic strategy for the treatment of ischemic heart failure.

DOI： 10.1016/j.jphs.2024.04.005

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DOI： 10.1007/s40830-024-00495-9

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While cysteine (CysSH) is known to be exported into the extracellular space, its biological significance is not well understood. The present study examined the movement of extracellular CysSH using stable isotope-labeled cystine (CysSSCys), which is transported into cells and reduced to CysSH. Exposure of HepG2 cells to 100 µM stable isotope-labeled CysSSCys resulted in 70 µM labeled CysSH in cell medium 1 h after CysSSCys exposure. When the cell medium was collected and incubated with either hydrogen peroxide (H2O2) or atmospheric electrophiles, such as 1,2-naphthoquinone, 1,4-naphthoquinone and 1,4-benzoquinone, CysSH in the cell medium was almost completely consumed. In contrast, extracellular levels of CysSH were unaltered during exposure of HepG2 cells to H2O2 for up to 2 h, suggesting redox cycling of CysSSCys/CysSH in the cell system. Experiments with and without changing cell medium containing CysSH from HepG2 cells revealed that oxidative and electrophilic modifications of cellular proteins, caused by exposure to H2O2 and 1,2-naphthoquinone, were significantly repressed by CysSH in the medium. We also examined participation of enzymes and/or antioxidants in intracellular reduction of CysSSCys to CysSH. These results provide new findings that extracellular CysSH derived from CysSSCys plays a role in the regulation of oxidative and electrophilic stress.

DOI： 10.1080/10715762.2024.2350524

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DOI： 10.1016/j.actamat.2024.119823

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Lipid droplet (LD) accumulation in hepatocytes is one of the major symptoms associated with fatty liver disease. Mitochondria play a key role in catabolizing fatty acids for energy production through β-oxidation. The interplay between mitochondria and LD assumes a crucial role in lipid metabolism, while it is obscure how mitochondrial morphology affects systemic lipid metabolism in the liver. We previously reported that cilnidipine, an already existing anti-hypertensive drug, can prevent pathological mitochondrial fission by inhibiting protein-protein interaction between dynamin-related protein 1 (Drp1) and filamin, an actin-binding protein. Here, we found that cilnidipine and its new dihydropyridine (DHP) derivative, 1,4-DHP, which lacks Ca2+ channel-blocking action of cilnidipine, prevent the palmitic acid-induced Drp1-filamin interaction, LD accumulation and cytotoxicity of human hepatic HepG2 cells. Cilnidipine and 1,4-DHP also suppressed the LD accumulation accompanied by reducing mitochondrial contact with LD in obese model and high-fat diet-fed mouse livers. These results propose that targeting the Drp1-filamin interaction become a new strategy for the prevention or treatment of fatty liver disease.

DOI： 10.3390/ijms25105446

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Publisher：株式会社医学書院  

DOI： 10.11477/mf.2425201829

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BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is associated with various neurological symptoms, including nausea, dizziness, headache, encephalitis, and epileptic seizures. SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood-brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the detailed mechanism has not been fully elucidated. METHODS: Using the original strain of SARS-CoV-2, the infection in BMECs was confirmed by a detection of intracellular RNA copy number and localization of viral particles. BMEC functions were evaluated by measuring transendothelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics, and expression levels of proinflammatory genes. BMEC signaling pathway was examined by comprehensive RNA-seq analysis. RESULTS: We observed that iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs) were infected with SARS-CoV-2. SARS-CoV-2 infection resulted in decreased TEER. In addition, SARS-CoV-2 infection decreased expression levels of tight junction markers CLDN3 and CLDN11. SARS-CoV-2 infection also increased expression levels of proinflammatory genes, which are known to be elevated in patients with COVID-19. Furthermore, RNA-seq analysis revealed that SARS-CoV-2 dysregulated the canonical Wnt signaling pathway in iPSC-BMELCs. Modulation of the Wnt signaling by CHIR99021 partially inhibited the infection and the subsequent inflammatory responses. CONCLUSION: These findings suggest that SARS-CoV-2 infection causes BBB dysfunction via Wnt signaling. Thus, iPSC-BMELCs are a useful in vitro model for elucidating COVID-19 neuropathology and drug development.

DOI： 10.1186/s12987-024-00533-9

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DOI： 10.1093/jb/mvae031

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Reactive sulfane sulfur species such as persulfides (RSSH) and H2S2 are important redox regulators and closely linked to H2S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H2S to HSOH, and then to H2S2 in the presence of excess H2S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H2S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H2S donors, the corresponding donors of H2S2 (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

DOI： 10.1038/s41467-024-46652-7

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DOI： 10.1038/s43247-024-01214-z

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We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.

DOI： 10.3390/ijms25042401

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Smoking is one of the most serious risk factors for cardiovascular diseases. Although cigarette mainstream and sidestream smoke are significant contributors to increased cardiovascular mortality and morbidity, the underlying mechanism is still unclear. Here, we report that exposure of rat neonatal cardiomyocytes to cigarette smoke extract (CSE) induces mitochondrial hyperfission-mediated myocardial senescence. CSE leads to mitochondrial fission and reactive oxygen species (ROS) production through the complex formation between mitochondrial fission factor Drp1 and actin-binding protein, filamin A. Pharmacological perturbation of interaction between Drp1 and filamin A by cilnidipine and gene knockdown of Drp1 or filamin A inhibited CSE-induced mitochondrial hyperfission and ROS production as well as myocardial senescence. We previously reported that Drp1 activity is controlled by supersulfide-induced Cys644 polysulfidation. The redox-sensitive Cys644 was critical for CSE-mediated interaction with filamin A. The administration of supersulfide donor, Na2S3 also improved mitochondrial hyperfission-mediated myocardial senescence induced by CSE. Our results suggest the important role of Drp1-filamin A complex formation on cigarette smoke-mediated cardiac risk and the contribution of supersulfide to mitochondrial fission-associated myocardial senescence.

DOI： 10.1016/j.jphs.2023.12.008

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Language：English   Publisher：(公社)日本薬理学会  

Language：English   Publisher：British Journal of Pharmacology  

Background and purpose: Maintaining mitochondrial quality is attracting attention as a new strategy to treat diabetes and diabetic complications. We previously reported that mitochondrial hyperfission by forming a protein complex between dynamin-related protein (Drp) 1 and filamin, mediates chronic heart failure and cilnidipine, initially developed as an L/N-type Ca2+ channel blocker, improves heart failure by inhibiting Drp1-filamin protein complex. We investigated whether cilnidipine improves hyperglycaemia of various diabetic mice models. Experimental Approach: Retrospective analysis focusing on haemoglobin A1c (HbA1c) was performed in hypertensive and hyperglycaemic patients taking cilnidipine and amlodipine. After developing diabetic mice by streptozotocin (STZ) treatment, an osmotic pump including drug was implanted intraperitoneally, followed by weekly measurements of blood glucose levels. Mitochondrial morphology was analysed by electron microscopy. A Ca2+ channel-insensitive cilnidipine derivative (1,4-dihydropyridine [DHP]) was synthesized and its pharmacological effect was evaluated using obese (ob/ob) mice fed with high-fat diet (HFD). Key Results: In patients, cilnidipine was superior to amlodipine in HbA1c lowering effect. Cilnidipine treatment improved systemic hyperglycaemia and mitochondrial morphological abnormalities in STZ-exposed mice, without lowering blood pressure. Cilnidipine failed to improve hyperglycaemia of ob/ob mice, with suppressing insulin secretion. 1,4-DHP improved hyperglycaemia and mitochondria abnormality in ob/ob mice fed HFD. 1,4-DHP and cilnidipine improved basal oxygen consumption rate of HepG2 cells cultured under 25 mM glucose. Conclusion and implications: Inhibition of Drp1-filamin protein complex formation becomes a new strategy for type 2 diabetes treatment.

DOI： 10.1111/bph.16487

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DOI： 10.1016/j.mtla.2023.101918

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Language：Japanese   Publisher：Japanese Society for Circulation Research  

DOI： 10.60370/jjcircres.46.3_17

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DOI： 10.1016/j.jallcom.2023.171470

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For decades, the major focus of redox biology has been oxygen, the most abundant element on Earth. Molecular oxygen functions as the final electron acceptor in the mitochondrial respiratory chain, contributing to energy production in aerobic organisms. In addition, oxygen-derived reactive oxygen species including hydrogen peroxide and nitrogen free radicals, such as superoxide, hydroxyl radical and nitric oxide radical, undergo a complicated sequence of electron transfer reactions with other biomolecules, which lead to their modified physiological functions and diverse biological and pathophysiological consequences (e.g. oxidative stress). What is now evident is that oxygen accounts for only a small number of redox reactions in organisms and knowledge of biological redox reactions is still quite limited. This article reviews a new aspects of redox biology which is governed by redox-active sulfur-containing molecules-supersulfides. We define the term 'supersulfides' as sulfur species with catenated sulfur atoms. Supersulfides were determined to be abundant in all organisms, but their redox biological properties have remained largely unexplored. In fact, the unique chemical properties of supersulfides permit them to be readily ionized or radicalized, thereby allowing supersulfides to actively participate in redox reactions and antioxidant responses in cells. Accumulating evidence has demonstrated that supersulfides are indispensable for fundamental biological processes such as energy production, nucleic acid metabolism, protein translation and others. Moreover, manipulation of supersulfide levels was beneficial for pathogenesis of various diseases. Thus, supersulfide biology has opened a new era of disease control that includes potential applications to clinical diagnosis, prevention and therapeutics of diseases.

DOI： 10.1111/bph.16271

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DOI： 10.1007/s40830-023-00458-6

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DOI： https://doi.org/10.1126/sciadv.adg8631

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Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S-nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

DOI： 10.1126/sciadv.adg8631

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Language：Japanese   Publisher：Japanese Society for Circulation Research  

DOI： 10.60370/jjcircres.46.2_1

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DOI： 10.1007/s10853-023-08707-w

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Language：Japanese   Publisher：日本心脈管作動物質学会  

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Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future.

DOI： 10.1038/s42003-023-04888-x

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DOI： 10.1016/j.jmps.2023.105277

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Language：Japanese   Publisher：日本脂質生化学会  

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Language：Others   Publisher：Cold Spring Harbor Laboratory  

The elucidation of gene-silencing mechanisms by RNA interference (RNAi) and antisense oligomers has drawn increasing attention to nucleic acid medicine. However, several challenges remain to be overcome, such as in vivo stability, target selectivity, drug delivery, and induced innate immunity. Here, we report a new, versatile, and highly-selective method to hack RNA by controlling RNA structure using short oligonucleotides (RNA hacking: RNAh) in living cells. The oligonucleotide, named Staple oligomer, hybridizes specifically to a target mRNA and artificially induces an RNA higher-order structure, RNA G-quadruplex (RGq), on the mRNA. As a result, the RGq allows effective suppression of the target protein translation. This technology does not require cooperation with bioprocesses including enzymatic reactions as in RNAi or antisense technologies, permitting the introduction of artificial nucleic acids into Staple oligomers to increase their in vivo stability without compromising their effectiveness. The method was validated by translational regulation of the mRNAs of TPM3, MYD88, and TRPC6, in a cell-free system and in living mammalian cells. In vivo application of the technology to TRPC6 mRNA allowed us to prevent cardiac hypertrophy in transverse aortic constriction (TAC)-treated mice with no detectable off-target effects. This technology provides new insights into gene therapy after RNAi and antisense technologies.

DOI： 10.1101/2023.04.18.537290

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Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.

DOI： 10.3390/ijms24043800

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DOI： 10.1016/j.actamat.2022.118588

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Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

DOI： 10.3390/md21010052

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Language：Japanese   Publisher：日本心脈管作動物質学会  

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Supersulfides play a role in redox homeostasis, but adaptive cell responses to excess sulfur stress are not well understood. To address this issue, we generated transgenic (Tg) mice overexpressing cystathionine gamma-lyase (CSE) to catalyze transformations of cystathionine to cysteine (CysSH) and of cystine (CysSSCys) to CysSH persulfide (CysSSH). CSE Tg mice treated with excess cystine exhibited clear sulfur stress, as manifested by atrophy of the organs. Contrary to expectations, CysSSH concentrations were comparable in a variety of tissues of wild-type (WT) and CSE Tg mice; however, plasma levels of CysSSH in CSE Tg mice were significantly higher than those in WT mice. This export of surplus intracellular CysSSH was also characterized in primary hepatocytes from CSE Tg mice. Exposure of primary hepatocytes to sodium tetrasulfide (Na₂S₄) as a sulfur stress generator resulted in enhancement and rapid decline of intracellular CysSSH levels, accompanied by a concentration-dependent release of CysSSH into the extracellular space. Interestingly, among all amino acids, CysSSCys was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during Na₂S₄ exposure, suggesting that cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout and overexpression of SLC7A11 and used them to confirm that SLC7A11 is a predominant antiporter of CysSSCys and CysSSH. Furthermore, we found that cellular proteins undergo polysulfidation in response to Na₂S₄-mediated sulfur stress, which results in a lower mitochondrial membrane potential and concentration-dependent Na₂S₄ cytotoxicity. These results suggest that there are CysSSCys-dependent antiporters that pump excess CysSSH from cells during sulfur stress.</p>

DOI： 10.14869/toxpt.50.1.0_p3-241

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>We are exposed to a variety of environmental factors in our daily lives. A wide variety of epidemiological studies have suggested that environmental electrophiles such as methylmercury (MeHg) and cigarette sidestream smoke (CSS) disrupt our homeostasis and increase the risk of cardiovascular disease. However, the underlying mechanism is unclear. We previously identified that aberrant activation of Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating protein, is a key determinant of cardiac remodeling and fragility. Here we show that supersulfides that are redox-active sulfur metabolites have a critical role in the mitochondrial quality and cardiac homeostasis and exposure to environmental electrophiles such as MeHg and CSS increased cardiac risk by disrupting supersulfide-mediated mitochondrial quality.</p><p>Supersulfides highly exist in healthy cardiomyocytes and maintained the proper mitochondrial fission and fusion balance by regulating Drp1 activity through Cys⁶⁴⁴ polysulfidation. In a failing heart, supersulfides were catabolized to hydrogen sulfides, and this change in sulfide metabolism promoted Cys⁶⁴⁴ depolysulfidation of Drp1, which led to dysfunctions of mitochondria and cardiomyocytes. Supersulfide administration reversed electrophile-evoked mitochondrial dysfunction and improved the cardiac function of heart failure model mice. These results suggest that depolysulfidation of Drp1 at Cys⁶⁴⁴ by environmental stress such as MeHg increases cardiac fragility.</p>

DOI： 10.14869/toxpt.50.1.0_s9-2

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Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

DOI： 10.3390/ijms24010102

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DOI： 10.1016/j.jmps.2022.105050

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DOI： 10.1016/j.jallcom.2022.165491

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Reactive sulfur species (RSS) play a role in redox homeostasis; however, adaptive cell responses to excessive intracellular RSS are not well understood. Therefore, in this study, we generated transgenic (Tg) mice overexpressing cystathionine gamma-lyase (CSE) to produce excessive RSS. Contrary to expectations, tissue concentrations of RSS, such as cysteine persulfide (CysSSH), were comparable in both wild-type and CSE Tg mice, but the plasma concentrations of CysSSH were significantly higher in CSE Tg mice than in wild-type mice. This export of surplus intracellular RSS was also observed in primary hepatocytes of CSE Tg mice. Exposure of primary hepatocytes to the RSS generator sodium tetrasulfide (Na2S4) resulted in an initial increase in the intracellular concentration of RSS, which later returned to basal levels after export into the extracellular space. Interestingly, among all amino acids, cystine (CysSSCys) was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during Na2S4 exposure, suggesting that the cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout and overexpression of SLC7A11 and used them to confirm SLC7A11 as the predominant antiporter of CysSSCys and CysSSH. We observed that the poor efflux of excess CysSSH from the cell enhanced cellular stresses induced by Na2S4 exposure, such as polysulfidation of intracellular proteins, mitochondrial damage, and cytotoxicity. These results suggest the presence of a cellular response to excess intracellular RSS that involves the extracellular efflux of excess CysSSH by a cystine-dependent transporter to maintain intracellular redox homeostasis.

DOI： 10.1016/j.redox.2022.102514

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Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates β-adrenoceptor (βAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated βAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting β-arrestin-mediated βAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure.

DOI： 10.1038/s41467-022-34194-9

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BACKGROUND AND PURPOSE: Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. EXPERIMENTAL APPROACH: Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. KEY RESULTS: Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. CONCLUSION AND IMPLICATIONS: Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation.

DOI： 10.1111/bph.15942

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Language：Japanese  

Gタンパク質共役型受容体(GPCRs)は,細胞内環境の変化(物理化学的刺激)を細胞内情報に変換し,伝達する上で極めて重要な役割を果たしている.リガンド刺激後,多くのGPCRはリン酸化され,β-アレスチン依存性の内在化によって再利用または分解される.このプロセスは,GPCRタンパク質の品質管理を維持するための重要な機構である.一方で,β-アレスチン感受性の低いGPCRがどのように品質管理されるかは不明であった.我々は,β-アレスチン低感受性のプリン作動性P2Y6受容体(P2Y6R)に着目し,リン酸化に依存しないGPCR内在化経路(Redox-dependent Alternative Internalization:REDAI)の存在を新たに見出した.P2Y6Rはマクロファージに高発現しており,大腸炎の発症・進展に深くかかわっている.我々は,食品中に含まれる親電子物質がP2Y6RのREDAIを誘導し,抗炎症効果をもたらす一方で,REDAIの抑制が大腸炎の悪化をもたらすことをマウスで実証した.これらの結果は,GPCRのREDAIを標的にする創薬が,炎症性疾患の画期的な治療戦略となることを強く示唆している.(著者抄録)

Language：Japanese   Publisher：(公社)日本薬理学会  

Gタンパク質共役型受容体(GPCRs)は,細胞内環境の変化(物理化学的刺激)を細胞内情報に変換し,伝達する上で極めて重要な役割を果たしている.リガンド刺激後,多くのGPCRはリン酸化され,β-アレスチン依存性の内在化によって再利用または分解される.このプロセスは,GPCRタンパク質の品質管理を維持するための重要な機構である.一方で,β-アレスチン感受性の低いGPCRがどのように品質管理されるかは不明であった.我々は,β-アレスチン低感受性のプリン作動性P2Y6受容体(P2Y6R)に着目し,リン酸化に依存しないGPCR内在化経路(Redox-dependent Alternative Internalization:REDAI)の存在を新たに見出した.P2Y6Rはマクロファージに高発現しており,大腸炎の発症・進展に深くかかわっている.我々は,食品中に含まれる親電子物質がP2Y6RのREDAIを誘導し,抗炎症効果をもたらす一方で,REDAIの抑制が大腸炎の悪化をもたらすことをマウスで実証した.これらの結果は,GPCRのREDAIを標的にする創薬が,炎症性疾患の画期的な治療戦略となることを強く示唆している.(著者抄録)

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2022&ichushi_jid=J01200&link_issn=&doc_id=20220908420014&doc_link_id=10.1254%2Ffpj.22045&url=https%3A%2F%2Fdoi.org%2F10.1254%2Ffpj.22045&type=Crossref&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00002_2.gif

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Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

DOI： 10.1016/j.isci.2022.104582

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Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure.

DOI： 10.1016/j.jphs.2022.04.007

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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

DOI： 10.1016/j.jphs.2022.04.010

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DOI： 10.1016/j.jphs.2022.04.010.

Language：English   Publisher：(公社)日本薬理学会  

ヒト人工多能性幹細胞(iPSC)由来小腸上皮細胞(iPSC-SIEC)が新型コロナウイルス(SARS-CoV-2)感染モデルとして使用できるか検討した。吸収細胞やパネト細胞などのiPSC-SIECをSARS-CoV-2に感染させレムデシビルで治療すると、iPSC-SIECの細胞内SARS-CoV-2複製が減少した。SARS-CoV-2感染はタイトジャンクション(TJ)指標のZO-3とCLDN1の発現レベルと、TJ動力学の完全性を評価する経上皮電気抵抗を低下させた。SARS-CoV-2感染は新型コロナウイルス感染症(COVID-19)患者で上昇する炎症性遺伝子の発現レベルを上昇させた。これらの結果から、iPSC-SIECはCOVID-19の病態と医薬品開発を解明するための有用なin vitroモデルであると考えられた。

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Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels.

DOI： 10.3390/cells11132041

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Language：Japanese   Publisher：日本心脈管作動物質学会  

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Language：English   Publisher：American Journal of Physiology - Renal Physiology  

Recent studies have revealed the connection between amino acid chirality and diseases. We have previously reported that the gut microbiota produces various D-amino acids in a murine acute kidney injury (AKI) model. Here, we further explored the pathophysiological role of D-alanine (D-Ala) in AKI. Levels of D-Ala were evaluated in a murine AKI model. We analyzed transcripts of the N-methyl-Daspartate (NMDA) receptor, a receptor for D-Ala, in tubular epithelial cells (TECs). The therapeutic effect of D-Ala was then assessed in vivo and in vitro. Finally, the plasma level of D-Ala was evaluated in patients with AKI. The Grin genes encoding NMDA receptor subtypes were expressed in TECs. Hypoxic conditions change the gene expression of Grin1, Grin2A, and Grin2B. D-Ala protected TECs from hypoxia-related cell injury and induced proliferation after hypoxia. These protective effects are associated with the chirality of D-Ala. D-Ala inhibits reactive oxygen species (ROS) production and improves mitochondrial membrane potential, through NMDA receptor signaling. The ratio of D-Ala to L-Ala was increased in feces, plasma, and urine after the induction of ischemia-reperfusion (I/R). Moreover, Enterobacteriaceae, such as Escherichia coli and Klebsiella oxytoca, produce D-Ala. Oral administration of D-Ala ameliorated kidney injury after the induction of I/R in mice. Deficiency of NMDA subunit NR1 in tubular cells worsened kidney damage in AKI. In addition, the plasma level of D-Ala was increased and reflected the level of renal function in patients with AKI. In conclusion, D-Ala has protective effects on I/R-induced kidney injury. Moreover, the plasma level of D-Ala reflects the estimated glomerular filtration rate in patients with AKI. D-Ala could be a promising therapeutic target and potential biomarker for AKI.

DOI： 10.1152/ajprenal.00198.2021

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DOI： 10.3390

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新型コロナウイルス感染症(COVID-19)は,2019年に中国・武漢で発症が確認されて以来,全世界で猛威を奮っている新興感染症である.有効な治療法は未だ確立されておらず,COVID-19重症化機構の解明,予防・治療法の確立が急務となっている.主な感染経路として,新型コロナウイルス(SARS-CoV-2)表面にあるspikeタンパク質(Sタンパク質)が宿主細胞膜上のSARS-CoV-2ウイルスの認識受容体angiotensin converting enzyme(ACE)2タンパク質と結合し,エンドサイトーシスを介して細胞内に侵入する.COVID-19は肺への重篤な障害が報告されているが,ACE2は肺だけではなく心臓や消化器など様々な組織に発現しているため,細胞間のウイルス感染拡大,つまり感染重症化は肺だけではなく全身の組織でも起こりうる.我々は,COVID-19重症化リスクを増加させる既往症に心疾患が含まれることや,COVID-19後遺症にも心機能障害が含まれることから,心臓でのSARS-CoV-2感染・重症化の機構に着目した.その結果,心臓のACE2受容体がCOVID-19重症化リスク因子と示唆されている様々な環境ストレス曝露によって増加すること,その分子機構として,心筋リモデリングを制御する膜タンパク質複合体(TRPC3-Nox2)形成が関与することを新たに見いだした.さらに,TRPC3-Nox2タンパク質複合体形成を阻害する既承認薬の中から,Sタンパク質曝露によるACE2内在化を抑制する化合物クロミプラミン(三環系抗うつ薬)を同定した.本稿では,心臓におけるTRPC3-Nox2複合体形成を介したACE2受容体の発現制御機構,および人工組換え三量体Sタンパク質を用いたinvitroスクリーニング(偽感染モデル)とその結果について紹介する.(著者抄録)

Language：English   Publishing type：Research paper (scientific journal)  

Although the number of patients with heart failure is increasing, a sufficient treatment agent has not been established. Oxidative stress and inflammation play important roles in the development of myocardial remodeling. When thioredoxin (Trx), an endogenous anti-oxidative and inflammatory modulator with a molecular weight of 12 kDa, is exogenously administered, it disappears rapidly from the blood circulation. In this study, we prepared a long-acting Trx, by fusing human Trx (HSA-Trx) with human serum albumin (HSA) and evaluated its efficacy in treating drug-induced heart failure. Drug-induced cardiomyopathy was created by intraperitoneally administering doxorubicin (Dox) to mice three times per week. A decrease in heart weight, increased myocardial fibrosis and markers for myocardial damage that were observed in the Dox group were suppressed by HSA-Trx administration. HSA-Trx also suppressed the expression of atrogin-1 and myostatin, myocardial atrophy factors in addition to suppressing oxidative stress and inflammation. In the Dox group, a decreased expression of endogenous Trx in cardiac tissue and an increased expression of macrophage migration inhibitory factor were observed, but these changes were restored to normal levels by HSA-Trx administration. These findings suggest that HSA-Trx improves the pathological condition associated with Dox-induced cardiomyopathy by its anti-oxidative/anti-inflammatory and myocardial atrophy inhibitory action.

DOI： 10.3390/pharmaceutics14030562

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Language：Japanese   Publisher：(公社)日本薬理学会  

新型コロナウイルス感染症(COVID-19)は,2019年に中国・武漢で発症が確認されて以来,全世界で猛威を奮っている新興感染症である.有効な治療法は未だ確立されておらず,COVID-19重症化機構の解明,予防・治療法の確立が急務となっている.主な感染経路として,新型コロナウイルス(SARS-CoV-2)表面にあるspikeタンパク質(Sタンパク質)が宿主細胞膜上のSARS-CoV-2ウイルスの認識受容体angiotensin converting enzyme(ACE)2タンパク質と結合し,エンドサイトーシスを介して細胞内に侵入する.COVID-19は肺への重篤な障害が報告されているが,ACE2は肺だけではなく心臓や消化器など様々な組織に発現しているため,細胞間のウイルス感染拡大,つまり感染重症化は肺だけではなく全身の組織でも起こりうる.我々は,COVID-19重症化リスクを増加させる既往症に心疾患が含まれることや,COVID-19後遺症にも心機能障害が含まれることから,心臓でのSARS-CoV-2感染・重症化の機構に着目した.その結果,心臓のACE2受容体がCOVID-19重症化リスク因子と示唆されている様々な環境ストレス曝露によって増加すること,その分子機構として,心筋リモデリングを制御する膜タンパク質複合体(TRPC3-Nox2)形成が関与することを新たに見いだした.さらに,TRPC3-Nox2タンパク質複合体形成を阻害する既承認薬の中から,Sタンパク質曝露によるACE2内在化を抑制する化合物クロミプラミン(三環系抗うつ薬)を同定した.本稿では,心臓におけるTRPC3-Nox2複合体形成を介したACE2受容体の発現制御機構,および人工組換え三量体Sタンパク質を用いたinvitroスクリーニング(偽感染モデル)とその結果について紹介する.(著者抄録)

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2022&ichushi_jid=J01200&link_issn=&doc_id=20220314400006&doc_link_id=%2Fcf4yakur%2F2022%2F015702%2F009%2F0119-0123%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fcf4yakur%2F2022%2F015702%2F009%2F0119-0123%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

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Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC.

DOI： 10.3390/ijms23041967

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Language：English   Publisher：Frontiers in Immunology  

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

DOI： 10.3389/fimmu.2021.803647

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN  

<p>The human myocardium contains robust cells that constantly beat from birth to death without being replaced, even when exposed to various environmental stresses. Myocardial robustness is thought to depend primarily on the strength of the reducing power to protect the heart from oxidative stress. Myocardial antioxidant systems are controlled by redox reactions, primarily via the redox reaction of Cys sulfhydryl groups, such as found in thioredoxin and glutathione. However, the specific molecular entities that regulate myocardial reducing power have long been debated. Recently, reactive sulfide species, with excellent electron transfer ability, consisting of a series of multiple sulfur atoms, i.e., Cys persulfide and Cys polysulfides, have been found to play an essential role in maintaining mitochondrial quality and function, as well as myocardial robustness. This review presents the latest findings on the molecular mechanisms underlying mitochondrial energy metabolism and the maintenance of quality control by reactive sulfide species and provides a new insight for the prevention of chronic heart failure.</p>

DOI： 10.3164/jcbn.21-84

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After ligand stimulation, many G protein–coupled receptors (GPCRs) undergo β-arrestin–dependent desensitization, during which they are internalized and either degraded or recycled to the plasma membrane. Some GPCRs are not subject to this type of desensitization because they lack the residues required to interact with β-arrestins. We identified a mechanism of redox-dependent alternative internalization (REDAI) that promotes the internalization and degradation of the purinergic P2Y6 receptor (P2Y6R). Synthetic and natural compounds containing electrophilic isothiocyanate groups covalently modified P2Y6R at Cys220, which promoted the ubiquitylation of Lys137 and receptor internalization and degradation in various mouse and human cultured cell lines. Endogenous electrophiles also promoted ligand-dependent P2Y6R internalization and degradation. P2Y6R is highly abundant in inflammatory cells and promotes the pathogenesis of colitis. Deficiency in P2Y6R protected mice against experimentally induced colitis, and mice expressing a form of P2Y6R in which Cys220 was mutated to nonmodifiable serine were more sensitive to the induction of colitis. Several other GPCRs, including A2BAR, contain cysteine and lysine residues at the appropriate positions to mediate REDAI, and isothiocyanate stimulated the internalization of A2BAR and of a form of P2Y2R with insertions of the appropriate residues. Thus, endogenous and exogenous electrophiles may limit colitis progression through cysteine modification of P2Y6R and may also mediate internalization of other GPCRs.

DOI： 10.1126/scisignal.abj0644

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Language：Japanese   Publisher：The Japanese Pharmacological Society  

DOI： 10.1254/fpj.22067

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Language：Japanese   Publisher：The Japanese Pharmacological Society  

DOI： 10.1254/fpj.21109

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Human iPS cell-derived cardiomyocyte (hiPSC-CM) is conceptually promising as an unlimited source of human cardiomyocytes for cardiac pharmacological assessment including pre-clinical safety testing. Currently, cardiomyocytes could be differentiated with high efficiency but remain immature in their structure and function, demonstrating fetal gene expression, disorganized morphology, and contractile functions that differ from those of adult cardiomyocytes. As such, pharmacological assessment using hiPSC-CMs may be inaccurate and of limited utility. As cardiomyocytes in a heart are regularly exposed to electrical stimulation and mechanical stress, bio-engineering approaches become more prevalent as a tool for hiPSC-CM maturation. However, the mechanisms by which electrical stimulation enhances cardiomyocyte maturation remain poorly explored. Visualization of sarcomere network immunostaining withα-actinin and motion field imaging of beating cardiomyocytes enabled us to quantitatively evaluate the structural and the functional maturation of a hiPSC-CM (iCell-cardiomyocyte², CDI Fujifilm) induced by continuous electrical stimulations. The electrical stimulation did not alter spontaneous activity of hiPSC-CM in a week, whereas it altered sarcomere length after 4-weeks electrical stimulation. Thus, the combined analysis showed that structural and functional maturation occur at different times, indicating distinct molecular mechanisms.</p>

DOI： 10.14869/toxpt.49.1.0_p-46s

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Language：Japanese   Publisher：The Academy of Pharmaceutical Science and Technology, Japan  

DOI： 10.14843/jpstj.82.15

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

Reactive sulfur species (RSS) exhibit high nucleophilicity/antioxidative property and thus is thought to play an important role in repressing electrophilic/oxidative stress. On the other hand, excess amount of RSS (sulfur stress) may disrupt redox homeostasis and increase health risk. Here, we examined adaptive response against intracellular sulfur stress. The present study suggests that there are cystine-dependent antiporters including SLC7A11 to excrete excess intracellular CysSSH.

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DOI： 10.2355/isijinternational.ISIJINT-2021-194

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Language：Japanese   Publisher：Japanese Pharmacological Society  

<p>Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. Effective treatments for COVID-19 have not yet been established. The main pathway of infection is that the Spike protein on the surface of SARS-CoV-2 binds to its recognition receptor, angiotensin converting enzyme (ACE) 2, on the host cell. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike protein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. These results provide the potentiality of clomipramine for improving COVID-19 aggravation and sequelae.</p>

DOI： 10.1254/jpssuppl.95.0_2-s15-3

CiNii Research

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Pharmacological Society  

<p>G protein-coupled receptors (GPCRs) play pivotal roles in converting physicochemical stimuli due to environmental changes to intracellular responses. After ligand stimulation, many GPCRs are desensitized and then recycled or degraded through phosphorylation and β-arrestin-dependent internalization, an important process to maintain protein quality control of GPCRs. However, it is unknown how GPCRs with low β-arrestin sensitivity are controlled. Here we unmasked a β-arrestin-independent GPCR internalization, named Redox-dependent Alternative Internalization (REDAI), focusing on β-arrestin-resistant purinergic P2Y₆ receptor (P2Y₆R). P2Y₆R is highly expressed in macrophage and pathologically contributes to the development of colitis in mice. Natural electrophiles including in functional foods induce REDAI-mediated P2Y₆R degradation leading to anti-inflammation in macrophages. Prevention of Cys220 modification on P2Y₆R resulted in aggravation of the colitis. These results strongly suggest that targeting REDAI on GPCRs will be a breakthrough strategy for the prevention and treatment of inflammatory diseases.</p>

DOI： 10.1254/fpj.22045

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PubMed

CiNii Research

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CiNii Research

Language：Japanese   Publisher：Japanese Pharmacological Society  

<p>G protein-coupled receptors (GPCRs) play pivotal roles in converting physicochemical stimuli due to environmental changes to intracellular responses. After ligand stimulation, many GPCRs are desensitized and then recycled or degraded through b-arrestin-dependent internalization, an important process to maintain protein quality control of GPCRs. However, it is unknown how GPCRs with low β-arrestin sensitivity are controlled. Here we unmasked a b-arrestin-independent GPCR internalization, named Redox-dependent alternative internalization (REDAI), using b-arrestin-resistant purinergic P2Y₆ receptor (P2Y₆R). Natural isothiocyanates (ITCs) covalently bind with Cys²²⁰ in the intracellular 3^rd loop of P2Y₆R, and promote internalization and degradation of P2Y₆R through ubiquitination of Lys¹³⁷ in the 2^nd loop. P2Y₆R is highly expressed in macrophage and pathologically contributes to the development of colitis in mice. Endogenous electrophiles, such as S-nitrosoglutathione, also induce P2Y₆R degradation leading to anti-inflammation in macrophages. Prevention of Cys²²⁰ modification on P2Y₆R resulted in aggravation of the colitis. Accordingly, targeting REDAI on GPCRs will be a breakthrough strategy for the prevention and treatment of inflammatory diseases.</p>

DOI： 10.1254/jpssuppl.95.0_1-s10-2

CiNii Research

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Pharmacological Society  

<p>Novel coronavirus infection disease 2019 (COVID-19) is an emerging infectious disease that has been rampant worldwide since its onset was confirmed in Wuhan, China in 2019. An effective therapy has not yet been established, and there is an urgent need to establish a breakthrough therapeutic strategy for the prevention and treatment of COVID-19 aggravation. The main route of infection is that the Spike protein (S protein) on the surface of SARS-CoV-2 binds to its recognition receptor, angiotensin converting enzyme (ACE) 2, on the host cell surface. Then, SARS-CoV-2 invades the cell via endocytosis-dependent pathway. Although the major symptom of COVID-19 is lung inflammation, ACE2 is expressed not only in the lungs but also in various tissues including heart and digestive organs. We focused on the molecular mechanism underlying the development of heart failure, a pathology involved in COVID-19 aggravation risk factors and COVID-19 squeals. We revealed that cardiac ACE2 receptors were upregulated by exposure to various environmental stresses reported as COVID-19 aggravation risk factors, and the formation of membrane protein complex between TRPC3 and NADPH oxidase (Nox) 2 that participates in myocardial remodeling underlies pathological ACE2 upregulation. Furthermore, we utilized the already approved drugs that inhibit TRPC3-Nox2 protein complex formation, and identified that clomipramine, a tricyclic antidepressant, has the best potency to suppress ACE2 internalization induced by S protein exposure. This review introduces the mechanism of pathological ACE2 receptor upregulation through TRPC3-Nox2 complex formation in the heart, and the identification of a breakthrough drug candidate using in vitro pseudo-infection screening system.</p>

DOI： 10.1254/fpj.21070

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CiNii Research

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Heart disease is a major aggravating symptom of the new coronavirus infection (COVID-19) and a major risk factor for COVID-19 aggravation. We previously reported that the membrane protein-protein interaction between transient receptor potential canonical (TRPC) 3 channel and NADPH oxidase 2 (Nox2) mediates the progression of heart failure with cardiac remodeling. Here, we found that the exposure of rodent cardiomyocytes to various COVID-19 aggravation risk factors, such as hyperglycemia, smoking, and anticancer agents, increased expression level of angiotensin converting enzyme (ACE) 2, by forming TRPC3-Nox2 protein complex. Pharmacological intervention of TRPC3-Nox2 complex formation suppressed not only cardiomyocyte atrophy but also increased ACE2 mRNA expression due to anticancer drug treatment. Therefore, we screened the top 13 approved drugs that can suppress TRPC3-Nox2 complex formation to identify a drug that can additionally suppress SARS-CoV-2-derived spike protein-induced ACE2 internalization. We found that the tricyclic antidepressant, clomipramine, potently suppressed SARS-CoV-2 infection of human iPS cell-derived cardiomyocytes, as well as cardiac contractile dysfunction and metabolic dysfunction. These results strongly suggest that risk factors for COVID-19 aggravation cause cardiac disorder through increased expression of ACE2, and that approved drugs that block this pathway can be indicated for the prevention and treatment of COVID-19 cardiac severity.</p>

DOI： 10.14869/toxpt.49.1.0_s23-1

CiNii Research

Language：Japanese   Publisher：Japanese Pharmacological Society  

<p>There are over 565 million people infected with COVID-19 and 6 million deaths in the world. All patients infected COVID-19 may experience post COVID-19 conditions. Conditions after COVID-19 infection have wide range symptoms lasting for 12 weeks or more. Most prevalent neurological and psychiatric symptoms were fatigue, cognitive dysfunction, depressive symptoms, and sleep disturbance. And patients with post COVID-19 conditions have an impairment of health-related quality of life. However, there is no specific treatment for this condition. At the conference, we will present the treatment of cognitive dysfunction and depressive symptoms in the patients with post COVID-19 conditions.</p>

DOI： 10.1254/jpssuppl.96.0_4-b-w16-2

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-021-26633-w

Language：Japanese  

心筋は,動物が生まれてから死ぬまで入れ替わることなく,常に拍動し続けるきわめて頑健な細胞である.最近の研究から,システインパースルフィド(CysSSH)やポリスルフィド(CysSnH)に代表される硫黄原子が複数連なった,電子授受能に優れた分子種(超硫黄分子)が,ミトコンドリアの形態機能維持を介して心筋のストレス抵抗力に寄与することが明らかになってきた.本稿では,超硫黄分子によるミトコンドリアのエネルギー代謝や品質管理維持の分子制御機構および心臓におけるその病態生理学的意義について,筆者らの最新の知見を紹介する.(著者抄録)

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-021-23363-x

Language：Japanese  

Language：Others   Publishing type：Research paper (scientific journal)  

Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind the CAP-induced structural perturbations of biomacromolecules. Here, we investigated the potential effects of CAP irradiation of amyloid β (Aβ), an amyloidogenic protein associated with Alzheimer’s disease. Using nuclear magnetic resonance spectroscopy, we observed gradual spectral changes in Aβ after a 10 s CAP pretreatment, which also suppressed its fibril formation, as revealed by thioflavin T assay. As per mass spectrometric analyses, these effects were attributed to selective oxidation of the methionine residue (Met) at position 35. Interestingly, this modification occurred when Aβ was dissolved into a pre-irradiated buffer, indicating that some reactive species oxidize the Met residue. Our results strongly suggest that the H2O2 generated in the solution by CAP irradiation is responsible for Met oxidation, which inhibits Aβ amyloid formation. The findings of the present study provide fundamental insights into plasma biology, giving clues for developing novel applications of CAP.

DOI： 10.3390/ijms22063116

Language：Japanese  

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DOI： 10.3999/jscpt.52.39

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3164/jcbn.68-1-Intro

Language：Japanese  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-020-70956-5

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jaci.2020.06.039

Language：Japanese  

Gタンパク質共役型受容体(GPCR)は、細胞外の環境変化に伴う物理化学的刺激を認識し、細胞内に適応するための情報を伝達する'センサー'タンパク質の1つである。GPCRは薬の最有力標的分子として世界中で研究され続けられているものの、われわれが普段から口にする食事がGPCRに及ぼす影響については、いまだ十分には解き明かされていない。本稿では、機能性食品成分によるGPCRのアティピカルな機能修飾から、食が自然治癒力を高める分子機構を読み解く。(著者抄録)

Language：English  

DOI： 10.2174/1874467213666200407090121

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1248/bpb.b20-00903

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-019-46252-2

Language：English   Publishing type：Research paper (scientific journal)  

Ibudilast attenuates doxorubicin-induced cytotoxicity by suppressing formation of TRPC3 channel and NADPH oxidase 2 protein complexes.
BACKGROUND AND PURPOSE: Doxorubicin is a highly effective anticancer agent but eventually induces cardiotoxicity associated with increased production of ROS. We previously reported that a pathological protein interaction between TRPC3 channels and NADPH oxidase 2 (Nox2) contributed to doxorubicin-induced cardiac atrophy in mice. Here we have investigated the effects of ibudilast, a drug already approved for clinical use and known to block doxorubicin-induced cytotoxicity, on the TRPC3-Nox2 complex. We specifically sought evidence that this drug attenuated doxorubicin-induced systemic tissue wasting in mice. EXPERIMENTAL APPROACH: We used the RAW264.7 macrophage cell line to screen 1,271 clinically approved chemical compounds, evaluating functional interactions between TRPC3 channels and Nox2, by measuring Nox2 protein stability and ROS production, with and without exposure to doxorubicin. In male C57BL/6 mice, samples of cardiac and gastrocnemius muscle were taken and analysed with morphometric, immunohistochemical, RT-PCR and western blot methods. In the passive smoking model, cells were exposed to DMEM containing cigarette sidestream smoke. KEY RESULTS: Ibudilast, an anti-asthmatic drug, attenuated ROS-mediated muscle toxicity induced by doxorubicin treatment or passive smoking, by inhibiting the functional interactions between TRPC3 channels and Nox2, without reducing TRPC3 channel activity. CONCLUSIONS AND IMPLICATIONS: These results indicate a common mechanism underlying induction of systemic tissue wasting by doxorubicin. They also suggest that ibudilast could be repurposed to prevent muscle toxicity caused by anticancer drugs or passive smoking.

DOI： 10.1111/bph.14777

Language：English  

TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN.
Vascular smooth muscle cells (VSMCs) play critical roles in the stability and tonic regulation of vascular homeostasis. VSMCs can switch back and forth between highly proliferative synthetic and fully differentiated contractile phenotypes in response to changes in the vessel environment. Although abnormal phenotypic switching of VSMCs is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty, how control of VSMC phenotypic switching is dysregulated in pathologic conditions remains obscure. We found that inhibition of canonical transient receptor potential 6 (TRPC6) channels facilitated contractile differentiation of VSMCs through plasma membrane hyperpolarization. TRPC6-deficient VSMCs exhibited more polarized resting membrane potentials and higher protein kinase B (Akt) activity than wild-type VSMCs in response to TGF-β1 stimulation. Ischemic stress elicited by oxygen-glucose deprivation suppressed TGF-β1-induced hyperpolarization and VSMC differentiation, but this effect was abolished by TRPC6 deletion. TRPC6-mediated Ca2+ influx and depolarization coordinately promoted the interaction of TRPC6 with lipid phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of Akt activation. Given the marked up-regulation of TRPC6 observed in vascular disorders, our findings suggest that attenuation of TRPC6 channel activity in pathologic VSMCs could be a rational strategy to maintain vascular quality control by fine-tuning of VSMC phenotypic switching.-Numaga-Tomita, T., Shimauchi, T., Oda, S., Tanaka, T., Nishiyama, K., Nishimura, A., Birnbaumer, L., Mori, Y., Nishida, M. TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN.

DOI： 10.1096/fj.201802811R

Language：English  

TRPC3-Nox2 axis mediates nutritional deficiency-induced cardiomyocyte atrophy.
Myocardial atrophy, characterized by the decreases in size and contractility of cardiomyocytes, is caused by severe malnutrition and/or mechanical unloading. Extracellular adenosine 5'-triphosphate (ATP), known as a danger signal, is recognized to negatively regulate cell volume. However, it is obscure whether extracellular ATP contributes to cardiomyocyte atrophy. Here, we report that ATP induces atrophy of neonatal rat cardiomyocytes (NRCMs) without cell death through P2Y2 receptors. ATP led to overproduction of reactive oxygen species (ROS) through increased amount of NADPH oxidase (Nox) 2 proteins, due to increased physical interaction between Nox2 and canonical transient receptor potential 3 (TRPC3). This ATP-mediated formation of TRPC3-Nox2 complex was also pathophysiologically involved in nutritional deficiency-induced NRCM atrophy. Strikingly, knockdown of either TRPC3 or Nox2 suppressed nutritional deficiency-induced ATP release, as well as ROS production and NRCM atrophy. Taken together, we propose that TRPC3-Nox2 axis, activated by extracellular ATP, is the key component that mediates nutritional deficiency-induced cardiomyocyte atrophy.

DOI： 10.1038/s41598-019-46252-2

Language：English   Publishing type：Research paper (scientific journal)  

Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload.
Chronic exposure to methylmercury (MeHg), an environmental electrophilic pollutant, reportedly increases the risk of human cardiac events. We report that exposure to a low, non-neurotoxic dose of MeHg precipitated heart failure induced by pressure overload in mice. Exposure to MeHg at 10 ppm did not induce weight loss typical of higher doses but caused mitochondrial hyperfission in myocardium through the activation of Drp1 by its guanine nucleotide exchange factor filamin-A. Treatment of neonatal rat cardiomyocytes with cilnidipine, an inhibitor of the interaction between Drp1 and filamin-A, suppressed mitochondrial hyperfission caused by low-dose MeHg exposure. Modification of cysteine residues in proteins with polysulfides is important for redox signaling and mitochondrial homeostasis in mammalian cells. We found that MeHg targeted rat Drp1 at Cys624, a redox-sensitive residue whose SH side chain forms a bulky and nucleophilic polysulfide (Cys624-S(n)H). MeHg exposure induced the depolysulfidation of Cys624-S(n)H in Drp1, which led to filamin-dependent activation of Drp1 and mitochondrial hyperfission. Treatment with NaHS, which acts as a donor for reactive polysulfides, reversed MeHg-evoked Drp1 depolysulfidation and vulnerability to mechanical load in rodent and human cardiomyocytes and mouse hearts. These results suggest that depolysulfidation of Drp1 at Cys624-S(n)H by low-dose MeHg increases cardiac fragility to mechanical load through filamin-dependent mitochondrial hyperfission.

DOI： 10.1126/scisignal.aaw1920

Language：English  

Language：English  

TRPC channels in exercise-mimetic therapy.
Physical exercise yields beneficial effects on all types of muscle cells, which are essential for the maintenance of cardiovascular homeostasis and good blood circulation. Daily moderate exercise increases systemic antioxidative capacity, which can lead to the prevention of the onset and progression of oxidative stress-related diseases. Therefore, exercise is now widely accepted as one of the best therapeutic strategies for the treatment of ischemic (hypoxic) diseases. Canonical transient receptor potential (TRPC) proteins are non-selective cation channels activated by mechanical stress and/or stimulation of phospholipase C-coupled surface receptors. TRPC channels, especially diacylglycerol-activated TRPC channels (TRPC3 and TRPC6; TRPC3/6), play a key role in the development of cardiovascular remodeling. We have recently found that physical interaction between TRPC3 and NADPH oxidase (Nox) 2 under hypoxic stress promotes Nox2-dependent reactive oxygen species (ROS) production and mediates rodent cardiac plasticity, and inhibition of the TRPC3-Nox2 protein complex results in enhancement of myocardial compliance and flexibility similar to that observed in exercise-treated hearts. In this review, we describe current understanding of the roles of TRPC channels in striated muscle (patho)physiology and propose that targeting TRPC-based protein complexes could be a new strategy to imitate exercise therapy.

DOI： 10.1007/s00424-018-2211-3

Language：English  

Mitochondrial dynamics in exercise physiology.
A growing body of evidence suggests that exercise shows pleiotropic effects on the maintenance of systemic homeostasis through mitochondria. Dysregulation of mitochondrial dynamism is associated with metabolic inflexibility, resulting in many of the metabolic diseases and aging. Studies have suggested that exercise prevents and delays the progression of mitochondrial dysfunction by improving mitochondrial metabolism, biogenesis, and quality control. Exercise modulates functions of mitochondrial dynamics-regulating proteins through post-translational modification mechanisms. In this review, we discuss the putative mechanisms underlying maintenance of mitochondrial homeostasis by exercise, especially focusing on the post-translational modifications of several signaling proteins contributing to mitochondrial biogenesis, autophagy or mitophagy flux, and fission/fusion cycle. We also introduce novel small molecules that can potentially mimic exercise therapy through preserving mitochondrial dynamism. These recent advancements in the field of mitochondrial biology may lead to a greater understanding of exercise signaling.

DOI： 10.1007/s00424-019-02258-3

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.RA118.006111

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DOI： 10.1007/s00424-019-02258-3

Language：English   Publishing type：Research paper (scientific journal)  

Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence.

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Mitochondria-specific SQR deficiency in mice causes lethal impairment of sulfur respiration

DOI： 10.1016/j.freeradbiomed.2018.10.209

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DOI： 10.3389/fphar.2018.00523

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DOI： 10.1016/j.bbrc.2017.12.088

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DOI： 10.1016/bs.ampbs.2018.01.002

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DOI： 10.1248/yakushi.18-00091-1

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DOI： 10.1248/yakushi.18-00091-F

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DOI： 10.1016/j.pharmthera.2017.06.010

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Role of TRPC3 and TRPC6 channels in the myocardial response to stretch: Linking physiology and pathophysiology.

DOI： 10.1016/j.pbiomolbio.2017.06.010

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-017-01311-y

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DOI： 10.1016/j.jphs.2017.09.002

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DOI： 10.1021/acs.chemrestox.7b00120

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DOI： 10.3389/fcvm.2017.00056

Language：English   Publishing type：Research paper (scientific journal)  

TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy
Myocardial atrophy is a wasting of cardiac muscle due to hemodynamic unloading. Doxorubicin is a highly effective anticancer agent but also induces myocardial atrophy through a largely unknown mechanism. Here, we demonstrate that inhibiting transient receptor potential canonical 3 (TRPC3) channels abolishes doxorubicin-induced myocardial atrophy in mice. Doxorubicin increased production of ROS in rodent cardiomyocytes through hypoxic stress-mediated upregulation of NADPH oxidase 2 (Nox2), which formed a stable complex with TRPC3. Cardiomyocyte-specific expression of TRPC3 C-terminal minipeptide inhibited TRPC3-Nox2 coupling and suppressed doxorubicin-induced reduction of myocardial cell size and left ventricular (LV) dysfunction, along with its upregulation of Nox2 and oxidative stress, without reducing hypoxic stress. Voluntary exercise, an effective treatment to prevent doxorubicin-induced cardiotoxicity, also downregulated the TRPC3-Nox2 complex and promoted volume load-induced LV compliance, as demonstrated in TRPC3-deficient hearts. These results illustrate the impact of TRPC3 on LV compliance and flexibility and, focusing on the TRPC3-Nox2 complex, provide a strategy for prevention of doxorubicin-induced cardiomyopathy.

DOI： 10.1172/jci.insight.93358

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DOI： 10.1016/j.freeradbiomed.2017.01.024

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DOI： 10.1038/s41598-017-07903-4

Language：Japanese  

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DOI： 10.1016/j.phrs.2017.03.013

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DOI： 10.3389/fphar.2017.00428

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DOI： 10.1161/JAHA.116.003601

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DOI： 10.1254/fpj.149.84

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TRPC3 participates in angiotensin II type 1 receptor-dependent stress-induced slow increase in intracellular Ca2+ concentration in mouse cardiomyocytes

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DOI： 10.1254/fpj.149.269

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DOI： 10.1038/srep39383

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DOI： 10.1038/srep37001

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DOI： 10.1038/srep28944

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DOI： 10.1016/j.abb.2015.11.008

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DOI： 10.3164/jcbn.15-111

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DOI： 10.1124/mol.115.102863

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DOI： 10.1126/scisignal.aac9187

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DOI： 10.1042/BJ20150596

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DOI： 10.1039/c6md00023a

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DOI： 10.1210/me.2015-1201

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DOI： 10.1074/jbc.M115.656710

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DOI： 10.1021/tx500416y

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Cardiac redox homeostasis is precisely regulated by reactive oxygen species (ROS) or electrophilic molecules that are formed by ROS reacting with intracellular substrates, and their eliminating systems. We have focused on the role of nitric oxide (NO) generated from inducible NO synthase (iNOS) that is continuously upregulated from early stage of heart failure, and revealed that iNOS-derived NO acts as a protective factor in the early stage of heart failure, whereas it contributes to induction of cardiac early senescence in later stages. The switching mechanism of NO-mediated signaling includes formation of endogenous NO-derived electrophilic byproducts such as 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), which selectively targets an oncogenic small GTPase H-Ras at Cys-184, leading to cardiac cell senescence via covalent modification (S-guanylation) and activation of H-Ras. We also found that hydrogen sulfide-related reactive sulfur species (RSS) function as potent nucleophiles to eliminate electrophilic modification of H-Ras and suppress the onset of chronic heart failure after myocardial infarction. Our results strongly suggest a new concept of redox biolog

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DOI： 10.1093/cvr/cvu185

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DOI： 10.1016/j.yjmcc.2014.02.003

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DOI： 10.1161/ATVBAHA.113.001974

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Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation.

DOI： 10.1371/journal.pone.0068351

Language：English   Publishing type：Research paper (scientific journal)  

Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice
N-type voltage-dependent Ca2+channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca2+ influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice. © 2013 Elsevier Inc.

DOI： 10.1016/j.bbrc.2013.03.040

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DOI： 10.1038/ncomms2540

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DOI： 10.1038/ncomms2540

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DOI： 10.1093/jb/mvs145

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DOI： 10.1074/jbc.M112.357871

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DOI： 10.1242/bio.20121370

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DOI： 10.1038/NCHEMBIO.1018

Language：English   Publishing type：Research paper (scientific journal)  

Recombinant mitochondrial transcription factor A protein inhibits nuclear factor of activated T cells signaling and attenuates pathological hypertrophy of cardiac myocytes.
The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy. (C) 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

DOI： 10.1016/j.mito.2012.06.002

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1161/ATVBAHA.110.221010

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1254/jphs.11R05CP

Language：English  

DOI： 10.1016/j.niox.2010.10.003

Language：English  

DOI： 10.1161/ATVBAHA.110.221010

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2011.04.124

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2011.04.124

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.1017640108

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1254/jphs.10279FP

Language：English   Publishing type：Research paper (scientific journal)  

Dual Signaling Pathways of Arterial Constriction by Extracellular Uridine 5 '-Triphosphate in the Rat
We investigated actions of uridine 5'-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 µM) elicited an inward-rectifying current strongly reminiscent of activation of P2X(1) receptor, and a similar current was also induced by α,β-methylene adenosine 5'-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X(1)-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 µM) elicited similar conductance single channel activities. UTP (≥10 µM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca(2+) or bath-applied 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 µM) or nifedipine (10 µM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 µM). Similar biphasic rises in [Ca(2+)](i) were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X(1) receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X(1)-like and P2Y receptors, but not TRPC3.

DOI： 10.1254/jphs.10281fp

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-1-61779-126-0_17

Language：Others  

Natriuretic Peptides-Guanylyl Cyclase-A Signaling Pathway Inhibits TRPC6-Mediated Pro-Hypertrophic Signaling in the Hearts

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1248/yakushi.130.1397

Language：Japanese  

DOI： 10.1248/yakushi.130.1427

Language：English   Publishing type：Research paper (scientific journal)  

Blockade of TRPC6 is a Novel Therapeutic Approach Against Pathological Cardiac Remodeling

Language：Japanese  

[Molecular mechanism underlying the development of heart failure mediated by heterotrimeric G protein signaling].

DOI： 10.1254/fpj.135.179

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M109.076232

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M109.074104

Language：English   Publishing type：Research paper (scientific journal)  

Ca2+ influx and protein scaffolding via TRPC3 sustain PKC beta and ERK activation in B cells

DOI： 10.1242/jcs.061051

Language：Japanese  

[Mechanism of cardiac hypertrophy via diacylglycerol-sensitive TRPC channels].
Activation of Ca²⁺ signaling in cardiomyocytes induced by receptor stimulation or mechanical stress has been implicated in the development of cardiac hypertrophy. However, it is still unclear how intracellular Ca²⁺ targets specifically decode the alteration of intracellular Ca²⁺ concentration ([Ca²⁺]_i) on the background of the rhythmic Ca²⁺ increases required for muscle contraction. In excitable cardiomyocytes, changes in the frequency or amplitude of Ca²⁺ transients evoked by Ca²⁺ influx-induced Ca²⁺ release have been suggested to encode signals for induction of hypertrophy, and a partial depolarization of plasma membrane by receptor stimulation will increase the frequency of Ca²⁺ oscillations. We found that activation of diacylglycerol (DAG)-responsive canonical transient receptor potential (TRPC) subfamily channels (TRPC3 and TRPC6) mediate membrane depolarization induced by G_q protein-coupled receptor stimulation. DAG-mediated membrane depolarization through activation of TRPC3/TRPC6 channels increases the frequency of Ca²⁺ spikes, leading to activation of calcineurin-dependent signaling pathways. Inhibition of either TRPC3 or TRPC6 completely suppressed agonist-induced hypertrophic responses, suggesting that TRPC3 and TRPC6 form heterotetramer channels. Furthermore, we found that hypertrophic agonists increase the expression of TRPC6 proteins through activation of G₁₂ family proteins, leading to amplification of DAG-mediated hypertrophic signaling in cardiomyocytes. As heart failure proceeds through cardiac hypertrophy, TRPC3/TRPC6 channels may be a new therapeutic target for heart failure.

DOI： 10.1248/yakushi.130.295

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/chin.201029249

Language：English   Publishing type：Research paper (scientific journal)  

Atrial and brain natriuretic peptides-guanylyl cyclase-A signaling pathway inhibits TRPC6-mediated pro-hypertrophic signaling in the hearts

Language：English   Publishing type：Research paper (scientific journal)  

Cardiac Natriuretic Peptides Inhibit TRPC6-Mediated Prohypertrophic Signaling

Language：Others  

Regulation of cardiac hypertrophy by the formation of G protein-coupled receptor--TRPC channel protein complex.

DOI： 10.1254/fpj.134.131

Language：English   Publishing type：Research paper (scientific journal)  

Amphotericin B-induced renal tubular cell injury is mediated by Na+ Influx through ion-permeable pores and subsequent activation of mitogen-activated protein kinasesand elevation of intracellular Ca2+ concentration.
Amphotericin B (AMB) is one of the most effective antifungal agents; however, its use is often limited by the occurrence of adverse events, especially nephrotoxicity. The present study was designed to determine the possible mechanisms underlying the nephrotoxic action of AMB. The exposure of a porcine proximal renal tubular cell line (LLC-PK1 cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, the leakage of lactate dehydrogenase, and tissue ATP depletion. Propidium iodide uptake was enhanced, while terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was not affected by AMB, suggesting a lack of involvement of apoptosis in AMB-induced cell injury. The cell injury was inhibited by the depletion of membrane cholesterol with methyl-beta-cyclodextrin, which lowered the extracellular Na(+) concentration or the chelation of intracellular Ca(2+). The rise in the intracellular Ca(2+) concentration may be mediated through the activation of the ryanodine receptor (RyR) on the endoplasmic reticulum and the mitochondrial Na(+)-Ca(2+) exchanger, since cell injury was attenuated by dantrolene (an RyR antagonist) and CGP37157 (an Na(+)-Ca(2+) exchange

DOI： 10.1128/AAC.01137-08

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/AAC.01137-08

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.0808793106

Language：English   Publishing type：Research paper (scientific journal)  

A food-derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor-initiated signal transduction

DOI： 10.1111/j.1471-4159.2008.05686.x

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/emboj.2008.237

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00210-008-0321-8

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/bi800199z

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M611780200

Language：English  

DOI： 10.1016/j.bbadis.2007.03.006

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/sj.emboj.7601417

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

TRP channels: Molecular diversity and physiological function

DOI： 10.1080/10739680600885111

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M602832200

Language：English   Publishing type：Research paper (scientific journal)  

Blocker-resistant presynaptic voltage-dependent Ca2+ channels underlying glutamate release in mice nucleus tractus solitarii

DOI： 10.1016/j.brainres.2006.05.077

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1161/01.RES.0000233356.10630.8a

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1254/jphs.FP0051036

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cellsig.2004.12.014

Language：English   Publishing type：Research paper (scientific journal)  

Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.

DOI： 10.1152/physiolgenomics.00229.2004

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jcb.M409397200

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M409710200

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/ng1393

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cγ2 (PLCγ2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCγ2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCγ2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCγ2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCγ2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCγ2. Strikingly, Ca2+ entry-activated PLCγ2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca 2+ entry with PLCγ2 translocation and activation controls the amplification and co-ordination of receptor signalling.

DOI： 10.1093/emboj/cdg457

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S0891-5849(03)00172-2

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1161/01.RES.0000043282.39776.7C

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1006/bbrc.2002.6553

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M107392200

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1084/jem.20011758

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S1097-2765(01)00438-5

Language：English   Publishing type：Research paper (scientific journal)  

Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS
however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the βγ-subunit of G protein (Gβγ) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The Gβγ-responsive ERK activation induced by H2O2 is independent of ligands binding to G(i)-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [35S]GTPγS binding to cardiac membranes and directly activates purified heterotrimeric G(i) and G(o) but not G(s). Analysis using heterotrimeric G(o) and its individual subunits indicates that H2O2 modifies Gα(o) but not Gβγ, which leads to subunit dissociation. We conclude that Gα(i) and Gα(o) are critical targets of oxidative stress for activation of ERK.

DOI： 10.1038/35044120

Event date： 2012.3

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Venue：京都   Country：Japan  

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Event date： 2010.3

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Venue：大阪   Country：Japan  

Event date： 2010.3

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Venue：京都大学桂キャンパス   Country：Japan  

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Event date： 2009.11

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Event date： 2009.9

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Venue：Joetsu Kokusai Ski Resort, Nagano   Country：Japan  

Event date： 2009.7

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Country：Japan  

Event date： 2009.7

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

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Venue：京都   Country：Japan  

Event date： 2009.3

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Venue：横浜   Country：Japan  

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Venue：神戸   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

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Event date： 2024.7

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Venue：イギリス   Country：Japan  

Event date： 2024.4

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Event date： 2023.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸国際会議場・神戸国際展示場2号館（神戸市）   Country：Japan  

Event date： 2023.12

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Country：Japan  

Event date： 2023.12

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Venue：神戸国際会議場・神戸国際展示場2号館   Country：Japan  

Event date： 2023.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸国際会議場・神戸国際展示場2号館（神戸市）   Country：Japan  

Event date： 2023.12

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.11

Language：Japanese  

Venue：第一薬科大学（福岡市南区）   Country：Japan  

Event date： 2023.10 - 2023.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡国際会議場・マリンメッセ福岡B館   Country：Japan  

Event date： 2023.10 - 2023.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡国際会議場・マリンメッセ福岡B館   Country：Japan  

Event date： 2023.10 - 2023.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡国際会議場・マリンメッセー福岡B館   Country：Japan  

Event date： 2023.10 - 2023.11

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Venue：福岡国際会議場・マリンメッセ福岡B館   Country：Japan  

Event date： 2023.10

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Country：Japan  

Event date： 2023.10

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Event date： 2023.10

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Venue：沖縄   Country：Japan  

Event date： 2023.10

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Venue：岡崎   Country：Japan  

Event date： 2023.10

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Venue：岡崎   Country：Japan  

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Venue：岡崎   Country：Japan  

Event date： 2023.9

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Venue：福岡   Country：Japan  

Event date： 2023.9

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Venue：福岡   Country：Japan  

Event date： 2023.9

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Venue：秋田   Country：Japan  

Event date： 2023.9

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Venue：岡山   Country：Japan  

Event date： 2023.8 - 2023.9

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Venue：秋田   Country：Japan  

Event date： 2023.7

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Event date： 2023.7

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Event date： 2023.7

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Venue：山形   Country：Japan  

Event date： 2023.6

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Event date： 2023.6

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Event date： 2023.6

Language：Japanese  

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Event date： 2023.6

Language：Japanese  

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Event date： 2023.6

Language：Japanese  

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Event date： 2023.6

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Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

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Event date： 2023.6

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Event date： 2023.6

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Venue：静岡   Country：Japan  

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Venue：静岡   Country：Japan  

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Event date： 2023.6

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Event date： 2023.6

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Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2023.6

Language：Japanese  

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Event date： 2023.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2023.6

Language：Japanese  

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Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2023.6

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2023.6

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Event date： 2023.6

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Venue：福岡   Country：Japan  

Event date： 2023.6

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Event date： 2023.6

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Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2023.5

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Venue：岡崎   Country：Japan  

Event date： 2023.5

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Event date： 2023.4

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Event date： 2023.2

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Event date： 2023.2

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