Authorship：Corresponding author   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   Publishing type：Research paper (scientific journal)  

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

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

The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR), promoted Ang II–induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II–induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein–dependent vascular hypertrophy but reduced β-arrestin–dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II–induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascular smooth muscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin–dependent proliferation to G protein–dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II.

DOI： doi: 10.1126/scisignal.aac9187.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y(6) receptor (P2Y(6)R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR), promoted Ang II-induced hypertension in mice. In mice, deletion of P2Y(6)R attenuated Ang II-induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y(6)R formed stable heterodimers, which enhanced G protein-dependent vascular hypertrophy but reduced beta-arrestin-dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y(6)R heterodimers by the P2Y(6)R antagonist MRS2578 suppressed Ang II-induced hypertension in mice. Furthermore, P2Y(6)R abundance increased with age in vascularsmoothmuscle cells. The increased abundance of P2Y(6)R converted AT1R-stimulated signaling in vascular smooth muscle cells from beta-arrestin-dependent proliferation to G protein-dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y(6)R heterodimers with age may increase the likelihood of hypertension induced by Ang II.

DOI： 10.1126/scisignal.aac9187

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS-) regulates the metabolism and signaling actions of various electrophiles. HS- reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS- biosynthesis. Cardiac HS-, in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS- on myocardial infarction-associated heart failure. Thus, this study reveals HS--induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.

DOI： 10.1038/NCHEMBIO.1018

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

DOI： 10.1073/pnas.1017640108

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)-beta. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (G alpha(12/13)) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF-beta, connective tissue growth factor, and periostin) and Ang-converting enzyme (ACE) was suppressed by the functional inhibition of G alpha(12/13). The expression of these fibrogenic genes through G alpha(12/13) by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G-protein-coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of G alpha(12/13) in cardiomyocytes by the extracellular nucleotides-stimulated P2Y(6) receptor triggers fibrosis in pressure overload-induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF-beta.

DOI： 10.1038/emboj.2008.237

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

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C(PLC)-mediated production of inositol-1,4,5-trisphosphate (IP3) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.

DOI： 10.1038/sj.emboj.7601417

Authorship：Lead author   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

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

DOI： 10.3390/cells14030216

PubMed

Language：Japanese   Publisher：The Japanese Pharmacological Society  

<p>Currently, a variety of anticancer agents are used in the treatment of cancer. Since anticancer agents are used continuously over a long time, they carry the risk of side effects. One of the major side effects is cardiac dysfunction. For example, doxorubicin, an anthracycline-type anticancer agent, is clinically restricted because of its dose-dependent cardiotoxicity. Cardiotoxicity includes decreased ejection fraction, arrhythmias, and congestive heart failure, all of which are associated with high mortality rates. Therefore, it is important to assess the risk of cardiotoxicity of anticancer agents in advance. Cardiomyocytes require energy to beat and retain an abundance of mitochondria. We established quantitative measurements of mitochondrial length and respiratory activities using cardiomyocytes. We found that exposure of human iPS cell-derived cardiomyocytes (hiPSC-CMs) to anticancer agents with reported cardiotoxicity enhanced mitochondrial hyperfission and the oxygen consumption rate was significantly reduced. Knockdown of dynamin-related protein 1 (Drp1), mitochondrial fission-accelerating GTP-binding protein, suppressed mitochondrial hyperfission in hiPSC-CMs. This indicates that visualizing mitochondrial functions in hiPSC-CMs will be helpful in assessing the risk of cardiotoxicity caused by anticancer agents and that maintaining mitochondrial quality will become a new strategy to reduce anticancer agents-induced cardiotoxicity. In this review, we present the evaluation of cardiotoxicity targeting mitochondrial quality in anticancer agents, using osimertinib, a non-small cell lung cancer drug, as an example.</p>

DOI： 10.1254/fpj.24056

Scopus

PubMed

CiNii Research

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

DOI： 10.1016/j.jphs.2024.07.007

Scopus

researchmap

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

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

The atrophic myocardium resulting from mechanical unloading and nutritional deprivation is considered crucial as maladaptive remodeling directly associated with heart failure, as well as interstitial fibrosis. Conversely, myocardial hypertrophy resulting from hemodynamic loading is perceived as compensatory stress adaptation. We previously reported the abundant presence of highly redox-active polysulfide molecules, termed supersulfide, with two or more sulfur atoms catenated in normal hearts, and the supersulfide catabolism in pathologic hearts after myocardial infarction correlated with worsened prognosis of heart failure. However, the impact of supersulfide on myocardial remodeling remains unclear. Here, we investigated the involvement of supersulfide metabolism in cardiomyocyte remodeling, using a model of adenosine 5′-triphosphate (ATP) receptor-stimulated atrophy and endothelin-1 receptor-stimulated hypertrophy in neonatal rat cardiomyocytes. Results revealed contrasting changes in intracellular supersulfide and its catabolite, hydrogen sulfide (H2S), between cardiomyocyte atrophy and hypertrophy. Stimulation of cardiomyocytes with ATP decreased supersulfide activity, while H2S accumulation itself did not affect cardiomyocyte atrophy. This supersulfide catabolism was also involved in myofibroblast formation of neonatal rat cardiac fibroblasts. Thus, unraveling supersulfide metabolism during myocardial remodeling may lead to the development of novel therapeutic strategies to improve heart failure.

DOI： 10.1016/j.jphs.2024.05.002

Web of Science

Scopus

PubMed

researchmap

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

Authorship：Corresponding author   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

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Free Radical Research  

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

Web of Science

Scopus

PubMed

researchmap

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

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

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Fluids and Barriers of the CNS  

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

Web of Science

Scopus

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English  

DOI： 10.1093/jb/mvae031

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Communications  

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

Web of Science

Scopus

PubMed

researchmap

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

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

Web of Science

Scopus

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Pharmacological Sciences  

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

Web of Science

Scopus

PubMed

researchmap

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

ラット新生仔の心筋細胞にタバコ煙抽出物(CSE)を曝露すると、ミトコンドリア(MT)過剰分裂を介して心筋細胞老化が誘発された。CSEはMT分裂因子Drp1とアクチン結合蛋白質であるフィラミンA(FIL-A)との複合体形成を介して、MT分裂と活性酸素種(ROS)産生を引き起こした。シルニジピンやDrp1またはFIL-Aの遺伝子ノックダウンによりDrp1とFIL-Aの相互作用が変化し、CSE誘発性MT過剰分裂とROS産生、および心筋細胞老化を阻害した。Drp1活性の中心である酸化還元感受性をもったCys644について、CSEを介したFIL-Aとの相互作用において重要な役割を果たしていた。超硫黄分子(SS)供与体Na2S3の投与により、CSE誘発性MT過剰分裂を介して心筋細胞老化を改善した。以上より、Drp1-FIL-A複合体形成がタバコ煙介在性心リスクにおいて重要な役割を果たしており、MT分裂関連心筋細胞老化に対するSSの寄与が考えられた。

Authorship：Last author,　Corresponding author   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

Web of Science

Scopus

PubMed

Authorship：Corresponding author   Language：English   Publisher： Int. J Mol Sci.  

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Cardiac remodeling involves compensatory alterations in heart mass, geometry, and function in response to hemodynamic stress or cardiac injury. Previous studies have attributed myocardial variations to the size of cardiomyocytes. Recently, supersulfide, a sulfur-catenated molecule, has emerged as a crucial regulator of cardiac robustness. Our earlier findings revealed a heightened presence of supersulfide in healthy mouse hearts, which undergoes catabolism to hydrogen sulfide (H2S) following myocardial infarction. Despite these observations, the precise role of supersulfide metabolism in governing cardiac cellular functions remains elusive. In this study, we use cardiomyocytes isolated from ventricular of neonatal rat cardiomyocytes (NRCMs). We found that supersulfide, but not H2S, positively regulates the size of cardiomyocytes. qPCR results elucidated supersulfide anabolism related gene, Solute carrier family 7 member 11 (Slc7A11), may have involved in regulating size of cardiomyocytes. Slc7A11 gene knockdown efficiently induced cardiomyocyte atrophy. These findings suggest that supersulfide plays a key role in regulating cardiac cell remodeling induced by receptor stimulation. The anabolism and catabolism of supersulfide in cardiac cells could provide a new strategy for the treatment of pathological cardiac remodeling.</p>

DOI： 10.14869/toxpt.51.1.0_p-12s

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Striated muscles, including cardiac muscle and red skeletal muscle which abundantly express myoglobin, play a crucial role not only in systemic motor function but also in the homeostasis of energy metabolism. The muscle atrophy that comes with age and disease will reduce systemic function. We have previously shown that the onset and progression of myocardial atrophy induced by the anticancer drug doxorubicin are mediated through the functional interactions between transient receptor potential canonical (TRPC) 3 protein and the ROS-producing NADPH oxidase 2 (Nox2), which is responsible for ROS production, on the cardiac myocyte membrane. We also identified ibudilast, a bronchodilator, can inhibit TRPC3-Nox2 complex formation and reduced the systemic muscle weight loss induced by doxorubicin. In this study, we investigated whether TRPC3/Nox2 complex formation is a therapeutic target for skeletal muscle atrophy in muscular dystrophy. In the skeletal muscle of Duchenne muscular dystrophy (mdx) model mice, TRPC3-Nox2 protein complex formation was markedly observed, and pharmacological inhibition of this complex formation by ibudilast attenuated skeletal muscle atrophy and motor functional loss in mdx mice. Furthermore, administration of pyrazole-3 (Pyr3), a TRPC3-selective inhibitor, to mdx mice similarly attenuated muscle atrophy and weakness. Oxidative stress caused in atrophied muscle cells was also suppressed by preventing TRPC3-Nox2 interaction. These results suggest that TRPC3-Nox2 complex formation may be a new therapeutic target for preventing myopathic muscle atrophy.</p>

DOI： 10.14869/toxpt.51.1.0_p-5s

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>The robustness of cardiomyocytes is supported by their superior redox homeostasis, and disruption of redox homeostasis leads to the onset and progression of cardiac disease. Supersulfides, which include catenated sulfur atoms (R-S_nSH) such as cysteine persulfide (Cys-SSH), have been recently identified as highly reactive sulfur metabolites and recognized as a key molecule to regulate redox homeostasis. This study aimed to elucidate the role of sulfur metabolism in maintaining cardiac robustness, and the impact of abnormal sulfur metabolism on ischemic and electrophilic stress-mediated cardiac dysfunction.</p><p>Supersulfides are reduced to hydrogen sulfides in cardiomyocytes after ischemic and electrophilic stress. This supersulfide catabolism decreased the contractile function of cardiomyocytes through mitochondrial hyperfission. We also found that the depletion of supersulfides promotes mitochondrial fission by decreasing polysulfidation of mitochondrial fission factor Drp1 at Cys644.</p><p>Our results suggest that the redox modification of Drp1 Cys644 has a pivotal role in the ischemic and electrophilic tolerance of cardiomyocytes. We found that this polysulfidated Cys644 is modified by S-glutathionylation. Drp1 was glutathionylated by oxidized GSSG but not reduced GSH. GSSG-mediated Drp1 glutathionylation protected cardiac function against ischemic and electrophilic stress in vitro and in vivo. These results suggest that sulfur metabolism has a pivotal role in ischemic and electrophilic stress-mediated cardiac dysfunction through Drp1 Cys644 redox modification.</p>

DOI： 10.14869/toxpt.51.1.0_s2-2

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Understanding individual differences, such as age and gender, is important to consider personalized medicine. Human iPS cells are expected to be used to understand individual differences. Previous studies reported that patient-derived iPS cells can predict responders and non-responders. Using a large-scale G-protein-coupled receptor screening and human iPS cell-based assays, we found that the COVID-19 drug remdesivir mediated urotensin-II receptor activation and resulted in drug-mediated cardiotoxicity. To understand the impact of genetic variance on the susceptibility to remdesivir–urotensin-II receptor signaling in humans, we analyzed single-nucleotide variant information from a large-scale genomic database from 14,000 Japanese individuals. We found that there are more than 2,000 variants in the urotensin-II receptor and 110 missense mutations involving a single amino acid substitution. In addition, we identified the mutation in the urotensin-II receptor gene are sensitive to remdesivir, which may possibly be more susceptible to urotensin-II receptor-mediated cardiotoxicity. These approach may be able to predict adverse drug responses in humans. </p><p>In the future, the development of a cell bank using patient-derived iPS cells would facilitate research on individual differences.</p>

DOI： 10.14869/toxpt.51.1.0_s30-5

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>Diabetes is a chronic metabolic disorder that affects nearly 10% of adult people worldwide. It is characterized by high levels of blood glucose, which can lead to a range of complications, such as cardiovascular disease, neuropathy, and retinopathy. Recently, mitochondrial quality control has been highlighted as a potential therapeutic target for treating diabetes and its complications. We previously reported that mitochondrial hyperfission by forming a protein complex between dynamin-related protein (Drp) 1 and filamin A (FLNa), mediates chronic heart failure and cilnidipine, developed as an L/N-type Ca²⁺ channel blocker, improves heart failure by inhibiting Drp1-FLNa protein complex. Therefore, we investigated whether cilnidipine improves hyperglycemia of various diabetic model mice.</p><p>Cilnidipine treatment improved systemic hyperglycemia and mitochondrial morphological abnormalities in STZ-exposed mice, without lowering blood pressure. In contrast, cilnidipine failed to improve hyperglycemia of <i> ob/ob</i> mice, by suppressing insulin secretion. Therefore, we have identified a Ca²⁺channel-insensitive cilnidipine derivative (1,4-DHP) that does not inhibit insulin release. 1,4-DHP improved hyperglycemia and mitochondria morphology abnormality in <i> ob/ob</i> mice fed high-fat diet. These results suggested that maintaining mitochondrial quality by inhibition of Drp1-FLNa becomes a new strategy for diabetes treatment to treat diabetes and diabetic complications.</p>

DOI： 10.14869/toxpt.51.1.0_s8-4

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>The number of cancer patients worldwide is increasing every year. On the other hand, there are still many unknowns regarding the development of cardiovascular disease in cancer patients. Osimertinib is used for treatment of epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer and significantly improves the prognosis. However, heart failure is observed in 1-5% of patients during treatment and QT prolongation in 10%, with a lethal course in some cases, but the mechanism of cardiotoxicity caused by osimertinib has not been elucidated.</p><p>In this study, we focused on the mitochondria of human iPS-derived cardiomyocytes (hiPSC-CMs) to analyze the cardiotoxicity mechanism of osimertinib. Exposure of hiPSC-CMs to anticancer drugs (osimertinib, doxorubicin, and trastuzumab) induced mitochondrial hyperfission and suppressed respiratory capacity. Our laboratory has previously found that sulfur metabolism plays an important role in maintaining mitochondrial quality in cardiomyocytes. When sulfur molecular donors (Na₂S, Na₂S₂, and Na₂S₃) were added, Na₂S most strongly inhibited osimertinib-induced mitochondrial dysfunction. Intracellular imaging using a sulfur molecular detection probe showed that osimertinib reduced intracellular H₂S concentrations, which were restored by Na₂S exposure.</p><p>These results indicate that osimertinib reduces intracellular H₂S levels concurrently with mitochondrial dysfunction in myocardium. Our results also indicate that maintenance of intracellular H₂S levels may contribute to the mitigation of osimertinib cardiotoxicity.</p>

DOI： 10.14869/toxpt.51.1.0_p-139

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>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.</p>

DOI： 10.14869/toxpt.51.1.0_p-147

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>A growing body of evidence suggests that transient receptor potential canonical (TRPC) 3 and 6 channels are involved in developing pathological remodeling of the heart. However, we found that activation of TRPC6 channel enhances β adrenoceptor (βAR)-stimulated myocardial positive inotropy and prevents chronic heart failure in mice by enhancing Zn²⁺ dynamics. This study aims to investigate whether TRPC6-mediated Zn²⁺ influx suppresses sympathetic overactivity-induced chronic heart failure in mice. </p><p>Chronic stimulation of βAR with isoproterenol (ISO; 30 mg/kg/day) for 4 weeks caused myocardial dysfunction in WT mice and Zn²⁺ permeation-dead (PD) TRPC6 mutant-expressing mice. Treatment with 2-[4-(2,3-dimethylphenyl)-piperazin-1-yl]-N- (2-ethoxyphenyl) acetamide (PPZ2), a TRPC3/6/7 channel activator, improved ISO-induced heart failure in WT mice but failed in TRPC6 (PD) mice. Zinpyr-1 imaging revealed that PPZ2 increased the intracellular Zn²⁺ pool in ISO-treated WT hearts, while this increase was not observed in ISO-treated TRPC6 (PD) hearts. In addition, the electrophysiological study demonstrated that TRPC6 (PD) mutant could permeate Na⁺, Ca²⁺ and K⁺ as much as WT, but failed to permeate Zn²⁺ after PPZ2 stimulation. PPZ2 improved ISO-induced impairment of L-type Ca²⁺ channel current. Furthermore, PPZ2 attenuated ISO-induced oxidative stress and supersulfide catabolism. In conclusion, these results suggest that activating TRPC6-mediated Zn²⁺ influx improves chronic heart failure by maintaining redox homeostasis.</p>

DOI： 10.14869/toxpt.51.1.0_p-4s

CiNii Research

Language：Japanese   Publisher：The Japanese Society of Toxicology  

<p>【Introduction】Advances such as mRNA vaccination against the SARS-CoV-2 have dramatically reduced the number of deaths from COVID-19. In contrast, only a few individuals developed myocarditis after mRNA vaccination. Therefore, there is concern about vaccine-induced cardiotoxicity. However, it remains unclear whether SARS-CoV-2 spike protein synthesized from mRNA or adjuvant in the vaccine has any effects on cardiomyocytes. In this study, we assessed the cardiac safety of spike protein and adjuvant using cardiotoxicity testings, such as human iPSC-derived cardiomyocytes (hiPSC-CMs).</p><p>【Methods】Recombinant SARS-CoV-2 spike protein derived from alpha and omicron strains was provided by Prof. Takahiro Kusakabe (Faculty of Agriculture, Kyushu University). Monophosphoryl lipid A was used as adjuvants. The hERG channel currents were measured by automated patch clamp using hERG-expressing cells. iCell cardiomyocytes 2.0 were used as hiPSC-CMs. Field potentials were recorded using a multi-electrode array system. </p><p>【Results】We investigated the effect of spike protein on hERG channel currents. The spike protein did not inhibit hERG channel currents even at concentrations 1000-fold higher than previously reported plasma spike protein. In addition, the spike protein did not prolong field potential duration or early after depolarization in hiPSC-CMs. Similar results were obtained with adjuvants.</p><p>【Conclusion】These results suggest that spike protein does not induce significant cardiac dysfunction. In the future, we are planning to examine the relationship between spike protein and adverse events, such as myocarditis, in more detail.</p>

DOI： 10.14869/toxpt.51.1.0_p-198

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：British Journal of Pharmacology  

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

Web of Science

Scopus

PubMed

researchmap

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

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

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

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

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

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

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

Web of Science

Scopus

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Marine Drugs  

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

Web of Science

Scopus

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

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

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Redox Biology  

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

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：British Journal of Pharmacology  

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

Web of Science

Scopus

PubMed

researchmap

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

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。

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cells  

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

Web of Science

Scopus

PubMed

researchmap

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

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

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

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

Web of Science

Scopus

PubMed

researchmap

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

Web of Science

Scopus

PubMed

Authorship：Corresponding author   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

Web of Science

PubMed

CiNii Research

researchmap

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

Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases.

DOI： 10.1038/s41467-021-26633-w

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

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

DOI： 10.1038/s41467-021-23363-x

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：English   Publishing type：Research paper (scientific journal)  

Background: Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism. Objective: We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions. Methods: To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch. Results: IP₃ receptor type 1 (IP₃R1) knockdown in astrocytes suppressed IL-6–induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IP₃R1-dependent astrocytic Ca²⁺ responses involved Ca²⁺ influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron–specific IL-6 knockdown, spinal astrocyte–specific IP₃R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch. Conclusion: Our findings suggest that IP₃R1/TRPC channel–mediated Ca²⁺ signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention.

DOI： 10.1016/j.jaci.2020.06.039

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y6 receptor (P2Y6R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y6R prevents or promotes heart failure. We demonstrate that inhibition of P2Y6R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y6R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y6R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y6R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y6R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y6R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y6R exacerbates pressure overload-induced heart failure in mice, although P2Y6R in cardiomyocytes contributes to the progression of cardiac fibrosis.

DOI： 10.1038/s41598-020-70956-5

Authorship：Last author,　Corresponding author   Language：English  

BACKGROUND: Transient receptor potential (TRP) channels, especially canonical TRP channel subfamily members 3 (TRPC3) and 6 (TRPC6), have attracted attention as a putative therapeutic target of heart | 1 failure. Moreover, TRPC3 and TRPC6 channels are physiologically important for maintaining cellular homeostasis. How TRPC3/C6 channels alter intracellular signaling from adaptation to maladaptation has been discussed for many years. We recently showed that formation of a protein signal complex between TRPC3 and NADPH oxidase (Nox) 2 caused by environmental stresses (e.g., hypoxia, nutritional deficiency, and anticancer drug treatment) promotes Nox2-dependent reactive oxygen species production and cardiac stiffness, including myocardial atrophy and interstitial fibrosis, in rodents. In fact, pharmacological prevention of the TRPC3-Nox2 protein complex can maintain cardiac flexibility in mice after anti-cancer drug treatment. CONCLUSION: In this mini-review, we discuss the relationship between TRPC3/C6 channels and cardiovascular disease, and propose a new therapeutic strategy by focusing on pathology-specific protein- protein interactions.

DOI： 10.2174/1874467213666200407090121

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Non-alcoholic steatohepatitis (NASH) is a disease that has progressed from non-alcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6 (TRPC3/6), reportedly participate in the development of fibrosis in cardiovascular and renal systems. We hypothesized that TRPC3/6 may also participate in NASH fibrosis. We evaluated the effects of TRPC3 or TRPC6 functional deficiency in a NASH mouse model using choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Wild-type (WT) and TRPC3 or TRPC6 gene-deficient (KO) mice were fed with CDAHFD or standard diet for 6 weeks. The CDAHFD-induced body weight loss in TRPC6 KO mice was significantly lower compared with WT mice with CDAHFD. CDAHFD treatment significantly increased TRPC3 mRNA expression level and tissue weight in WT liver, which were suppressed in TRPC3 KO mice. However, either systemic deletion of TRPC3 or TRPC6 failed to attenuate liver steatosis, inflammation and fibrosis. These results imply that TRPC3 and TRPC6 are unlikely to be involved in liver dysfunction and fibrosis of NASH model mice.

DOI： 10.1248/bpb.b20-00903

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

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

Authorship：Last author,　Corresponding author   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

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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   Publishing type：Research paper (scientific journal)  

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)  

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC- electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H
₂
O
₂
exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H
₂
O
₂
cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

DOI： 10.1074/jbc.RA118.006111

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

Prolonged stimulation of β2-adrenergic receptor with β2-agonists impairs insulin actions in H9c2 cells.

Language：English   Publishing type：Research paper (conference, symposium, etc.)  

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

DOI： 10.1016/j.freeradbiomed.2018.10.209

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca²⁺-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca²⁺-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca²⁺ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca²⁺/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP₃-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

DOI： 10.3389/fphar.2018.00523

Language：English   Publishing type：Research paper (conference, symposium, etc.)  

Cysteine hydropersulphide (CysSSH) is a cysteine derivative having one additional sulphur atom bound to a cysteinyl thiol group. Recent advances in the development of analytical methods for detection and quantification of persulphides and polysulphides have revealed the biological presence, in both prokaryotes and eukaryotes, of hydropersulphides in diverse forms such as CysSSH, homocysteine hydropersulphide, glutathione hydropersulphide, bacillithiol hydropersulphide, coenzyme A hydropersulphide, and protein hydropersulphides. Owing to the chemical reactivity of the persulphide moiety, biological systems utilize persulphides as important intermediates in the synthesis of various sulphur-containing biomolecules. Accumulating evidence has revealed another important feature of persulphides: their potent reducing activity, which implies that they are implicated in the regulation of redox signalling and antioxidant functions. In this chapter, we discuss the biological occurrence and possible biosynthetic mechanisms of CysSSH and related persulphides, and we include descriptions of recent advances in the analytical methods that have been used to detect and quantitate persulphide species. We also discuss the antioxidant activity of persulphide species that contributes to protecting cells from reactive oxygen species-associated damage, and we examine the signalling roles of CysSSH in bacteria.

DOI： 10.1016/bs.ampbs.2018.01.002

Language：English   Publishing type：Research paper (conference, symposium, etc.)  

Moderate exercise has been reported to combat several diseases, including cardiovascular diseases and depressants. However, many patients do not have ability to undergo exercise therapy due to aging and severity of the symptoms. Therefore development of new drugs that can imitate exercise therapy is desired and actually studied worldwide. The heart is one of the physical load-responsive target organs such as skeletal muscles and vascular smooth muscles. The heart can adapt from environmental stress by changing its structure and morphology (i.e., remodeling). Physiological remodeling, caused by exercise or pregnancy, can be defined by compensative and reversible changes to the heart, whereas pathological remodeling can be defined by irreversible changes of the heart, through aberrant calcium ion (Ca
<sup>2

＋</sup>
) signaling as well as production of reactive oxygen species (ROS). However, crosstalk between Ca
<sup>2

＋</sup>
and ROS remains obscure. In this review we will introduce our recent findings on the functional crosstalk between transient receptor potential canonical (TRPC) 3 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) 2 as a novel molecular target to mimic exercise therapy.

DOI： 10.1248/yakushi.18-00091-1

Language：English   Publishing type：Research paper (conference, symposium, etc.)  

DOI： 10.1248/yakushi.18-00091-F

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

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP+) treatment (a model of Parkinson's disease). We show that MPP+-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12 cells (NPC12 cells) and rat cerebellar granule neurons (CGNs). Following MPP+ treatment, we found production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12 cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP+ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP+-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP+-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP+-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.

DOI： 10.1016/j.bbrc.2017.12.088

Authorship：Last author,　Corresponding author   Language：English  

Purinergic signaling, mediated mainly by G protein-coupled"P2Y receptors (P2YRs), is now attracting attention as a new therapeutic target for preventing or treating cardiovascular diseases. Observations using mice with-genetically modified P2YRs and/or treated with a pharmacological P2YR inhibitor have helped us understand the physiological and pathological significance of P2YRs in the cardiovascular system. P2YR-mediated biological functions are predominantly activated by mononucleotides released from non-adrenergic, non-cholinergic nerve endings or non-secretory tissues in response to physical stress or cell injury, though recent studies have suggested the occurrence of ligand-independent P2YR function through receptor-receptor interactions (oligomerization) in several biological processes. In this review, we introduce the functions of P2YRs and possible dimerization with G protein-coupled receptors (GPCRs) in the cardiovascular system. We focus especially on the crosstalk between uridine nucleotide-responsive P2Y(6)R and angiotensin (Ang) II typel receptor (AT1R) signaling, and introduce our recent finding that the P2Y(6)R antagonist MRS2578 interrupts heterodimerization between P2Y(6)R and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2Y(6)R oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases. (C) 2017 The Author(s). Published by Elsevier Inc.

DOI： 10.1016/j.pharmthera.2017.06.010

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

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)  

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

DOI： 10.1038/s41467-017-01311-y

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

The kidney expresses protease-activated receptor-1 (PAR-1). PAR-1 is known as a thrombin receptor, but its role in kidney injury is not well understood. In this study, we examined the contribution of PAR-1 to kidney glomerular injury and the effects of its inhibition on development of nephropathy. Mice were divided into 3 groups: control, doxorubicin + vehicle (15 mg/kg doxorubicin and saline) and doxorubicin + Q94 (doxorubicin at 15 mg/kg and the PAR-1 antagonist Q94 at 5 mg/kg/d) groups. Where indicated, doxorubicin was administered intravenously and PAR-1 antagonist or saline vehicle by subcutaneous osmotic mini-pump. PAR-1 expression was increased in glomeruli of mice treated with doxorubicin. Q94 treatment significantly suppressed the increased albuminuria in these nephropathic mice. Pathological analysis showed that Q94 treatment significantly attenuated periodic acid-Schiff and desmin staining, indicators of podocyte injury, and also decreased glomerular levels of podocin and nephrin. Furthermore, thrombin increased intracellular calcium levels in podocytes. This increase was suppressed by Q94 and Rox4560, a transient receptor potential cation channel (TRPC) 3/6 antagonist. In addition, both Q94 and Rox4560 suppressed the doxorubicin-induced increase in activities of caspase-9 and caspase-3 in podocytes. These data suggested that PAR-1 contributes to development of podocyte and glomerular injury and that PAR-1 antagonists have therapeutic potential. (C) 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.

DOI： 10.1016/j.jphs.2017.09.002

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

Electrophiles such as methylmercury (MeHg) affect, cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.

DOI： 10.1021/acs.chemrestox.7b00120

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cardiac stiffness, caused by interstitial fibrosis due to deposition of extracellular matrix proteins, is thought as a major clinical outcome of heart failure with preserved ejection fraction (HFpEF). Canonical transient receptor potential (TRPC) subfamily proteins are components of Ca²⁺-permeable non-selective cation channels activated by receptor stimulation and mechanical stress, and have been attracted attention as a key mediator of maladaptive cardiac remodeling. How TRPC-mediated local Ca²⁺ influx encodes a specific signal to induce maladaptive cardiac remodeling has been long obscure, but our recent studies suggest a pathophysiological significance of channel activity-independent function of TRPC proteins for amplifying redox signaling in heart. This review introduces the current understanding of the physiological and pathophysiological roles of TRPCs, especially focuses on the role of TRPC3 as a positive regulator of reactive oxygen species (PRROS) in heart. We have revealed that TRPC3 stabilizes NADPH oxidase 2 (Nox2), a membrane-bound reactive oxygen species (ROS)-generating enzyme, by forming stable protein complex with Nox2, which leads to amplification of mechanical stress-induced ROS signaling in cardiomyocytes, resulting in induction of fibrotic responses in cardiomyocytes and cardiac fibroblasts. Thus, the TRPC3 function as PRROS will offer a new therapeutic strategy for the prevention or treatment of HFpEF.

DOI： 10.3389/fcvm.2017.00056

Authorship：Last author,　Corresponding author   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

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Maintaining a redox balance by means of precisely controlled systems that regulate production, and elimination, and metabolism of electrophilic substances (electrophiles) is essential for normal cardiovascular function. Electrophilic signaling is mainly regulated by endogenous electrophiles that are generated from reactive oxygen species, nitric oxide, and the derivative reactive species of nitric oxide during stress responses, as well as by exogenous electrophiles including compounds in foods and environmental pollutants. Among electrophiles formed endogenously, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) has unique cell signaling functions, and pathways for its biosynthesis, signaling mechanism, and metabolism in cells have been clarified. Reactive persulfide species such as cysteine persulfides and polysulfides that are endogenously produced in cells are likely to be involved in 8-nitro-cGMP metabolism. These new aspects of redox biology may stimulate innovative and multidisciplinary research in cardiovascular physiology and pathophysiology. In our review, we focus on the redox-dependent regulation of electrophilic signaling via reduction and metabolism of electrophiles by reactive persulfides in cardiac cells, and we include suggestions for a new therapeutic strategy for cardiovascular disease.

DOI： 10.1016/j.freeradbiomed.2017.01.024

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.

DOI： 10.1038/s41598-017-07903-4

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Aging has a remarkable effect on cardiovascular homeostasis and it is known as the major non-modifiable risk factor in the development of hypertension. Medications targeting sympathetic nerve system and/or renin-angiotensin-aldosterone system are widely accepted as a powerful therapeutic strategy to improve hypertension, although the control rates remain unsatisfactory especially in the elder patients with hypertension. Purinergic receptors, activated by adenine, uridine nucleotides and nucleotide sugars, play pivotal roles in many biological processes, including platelet aggregation, neurotransmission and hormone release, and regulation of cardiovascular contractility. Since clopidogrel, a selective inhibitor of G protein-coupled purinergic P2Y(12) receptor (P2Y(12)R), achieved clinical success as an anti-platelet drug, P2YRs has been attracted more attention as new therapeutic targets of cardiovascular diseases. We have revealed that UDP-responsive P2Y(6)R promoted angiotensin type 1 receptor (AT1R)-stimulated vascular remodeling in mice, in an age-dependent manner. Moreover, the age-related formation of heterodimer between AT1R and P2Y(6)R was disrupted by MRS2578, a P2Y(6)R-selective inhibitor. These findings suggest that P2Y(6)R is a therapeutic target to prevent age-related hypertension. (C) 2017 Published by Elsevier Ltd.

DOI： 10.1016/j.phrs.2017.03.013

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

Stimulation of Adenosine A(2B) Receptor Inhibits Endothelin-1-Induced Cardiac Fibroblast Proliferation and alpha-Smooth Muscle Actin Synthesis Through the cAMP/Epac/PI3K/Akt-Signaling Pathway
Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express alpha-smooth muscle actin (alpha-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and alpha-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A(2) receptor inhibit ET-1-induced fibroblast proliferation and alpha-SMA expression in cardiac fibroblasts are not clearly identified.
Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of alpha-SMA.
Key results: Stimulation of adenosine subtype 2B (A(2B)) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced alpha-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A(2B) receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation.
Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A(2B) receptor. Thus, A(2B) receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis.

DOI： 10.3389/fphar.2017.00428

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

Background-Recent studies have shown that plasma levels of the biologically inactive prohormone for brain natriuretic peptide (proBNP) are increased in patients with heart failure. This can contribute to a reduction in the effectiveness of circulating BNP and exacerbate heart failure progression. The precise mechanisms governing the increase in proBNP remain unclear, however.
Methods and Results-We used our recently developed, highly sensitive human proBNP assay system to investigate the mechanisms underlying the increase in plasma proBNP levels. We divided 53 consecutive patients hospitalized with heart failure into 2 groups based on their aortic plasma levels of immunoreactive BNP. Patients with higher levels exhibited more severe heart failure, a higher proportion of proBNP among the immunoreactive BNP forms secreted from failing hearts, and a weaker effect of BNP as estimated from the ratio of plasma cyclic guanosine monophosphate levels to log-transformed plasma BNP levels. Glycosylation at threonines 48 and 71 of human proBNP contributed to the increased secretion of proBNP by attenuating its processing, and GalNAc-transferase (GALNT) 1 and 2 mediated the glycosylation-regulated increase in cardiac human proBNP secretion. Cardiac GALNT1 and 2 expression was suppressed by microRNA (miR)-30, which is abundantly expressed in the myocardium of healthy hearts, but is suppressed in failing hearts.
Conclusions-We have elucidated a novel miR-30-GALNT1/2 axis whose dysregulation increases the proportion of inactive proBNP secreted by the heart and impairs the compensatory actions of BNP during the progression of heart failure.

DOI： 10.1161/JAHA.116.003601

Language：English   Publishing type：Research paper (conference, symposium, etc.)  

DOI： 10.1254/fpj.149.84

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

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

Language：English   Publishing type：Research paper (conference, symposium, etc.)  

DOI： 10.1254/fpj.149.269

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

Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF)beta stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubuleassociated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation.

DOI： 10.1038/srep39383

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

Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.

DOI： 10.1038/srep37001

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

Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as "S-mercuration", potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 mu M MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

DOI： 10.1038/srep28944

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

Reactive oxygen (oxidant) and free radical species are known to cause nonspecific damage of various biological molecules. The oxidant toxicology is developing an emerging concept of the physiological functions of reactive oxygen species in cell signaling regulation. Redox signaling is precisely modulated by endogenous electrophilic substances that are generated from reactive oxygen species during cellular oxidative stress responses. Among diverse electrophilic molecular species that are endogenously generated, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a unique second messenger whose formation, signaling, and metabolism in cells was recently clarified. Most important, our current studies revealed that reactive cysteine persulfides that are formed abundantly in cells are critically involved in the metabolism of 8-nitro-cGMP. Modern redox biology involves frontiers of cell research and stem cell research; medical and clinical investigations of infections, cancer, metabolic syndrome, aging, and neurodegenerative diseases; and other fields. 8-Nitro-cGMP-mediated signaling and metabolism in cells may therefore be potential targets for drug development, which may lead to discovery of new therapeutic agents for many diseases.

DOI： 10.1016/j.abb.2015.11.008

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

Redox signaling is a key modulator of oxidative stress induced by nonspecific insults of biological molecules generated by reactive oxygen species. Current redox biology is revisiting the traditional concept of oxidative stress, such that toxic effects of reactive oxygen species are protected by diverse antioxidant systems upregulated by oxidative stress responses that are physiologically mediated by redox-dependent cell signaling pathways. Redox signaling is thus precisely regulated by endogenous electrophilic substances that are generated from reactive oxygen species and nitric oxide and its derivative reactive species during stress responses. Among electrophiles formed endogenously, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) has unique cell signaling functions, and pathways for its biosynthesis, signaling mechanism, and metabolism in cells have been clarified. Reactive sulfur species such as cysteine hydropersulfides that are abundant in cells are likely involved in 8-nitro-cGMP metabolism. These new aspects of redox biology may stimulate innovative and multidisciplinary research in cell and stem cell biology; infectious diseases, cancer, metabolic syndrome, ageing, and neurodegenerative diseases; and other oxidative stress-related disorders. This review focuses on the most recent progress in the biosynthesis, cell signaling, and metabolism of 8-nitro-cGMP, which is a likely target for drug development and lead to discovery of novel therapeutics for many diseases.

DOI： 10.3164/jcbn.15-111

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

Transient receptor potential canonical (TRPC) proteins form Ca2+-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl) piperazin-1-yl](3-fluorophenyl) methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)-piperazin-1-yl]-N-(2-ethoxyphenyl) acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca2+ influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca2+ signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca2+-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca2+ signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

DOI： 10.1124/mol.115.102863

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of beta-adrenergic receptors (beta ARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained beta AR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained beta AR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT) 4 expression that were mediated by the beta(2)AR subtype in cardiomyocytes and heart tissue. Overstimulation of beta(2)AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, beta AR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with beta-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in beta AR-mediated insulin resistance.

DOI： 10.1210/me.2015-1201

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca2+ influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes.

DOI： 10.1042/BJ20150596

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

The transient receptor potential canonical 3 (TRPC3) channel is a member of the TRPC family that contributes to the entry of Ca²⁺ through the plasma membrane or modulates the driving force for Ca²⁺ entry channels. The pyrazole compound Pyr3 has recently been reported to be a selective TRPC3 inhibitor and has become an attractive research tool and therapeutic agent for the treatment of heart failure. However, the in vivo characteristics of Pyr3 have not been investigated. To monitor the fate of Pyr3 in vivo, we designed and synthesized a radioiodinated Pyr3 probe ([¹²⁵I]I-Pyr3) by introducing radioiodine at the 2-position of the central phenyl ring of Pyr3. I-Pyr3 was shown to have direct TRPC3 inhibition activity similar to that of Pyr3 in TRPC3-overexpressing HEK293 cells. Using the tributyltin derivative as a radioiodination precursor, [¹²⁵I]I-Pyr3 was successfully prepared with high radiochemical purity. Biodistribution studies of [¹²⁵I]I-Pyr3 and [¹²⁵I]I-Pyr8 (the esterolysis product of [¹²⁵I]I-Pyr3) indicated high uptake of intact [¹²⁵I]I-Pyr3 in the lung and rapid metabolism to [¹²⁵I]I-Pyr8. These findings provide useful information about the in vivo kinetics of the selective TRPC inhibitor Pyr3.

DOI： 10.1039/c6md00023a

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

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.

DOI： 10.1074/jbc.M115.656710

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

Sulfhydration by a hydrogen sulfide anion and electrophile thiolation by reactive sulfur species (RSS) such as persulfides/polysulfides (e.g., R-S-SH/R-S-S-n-H(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH(2)-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH(2)-SH) and a generator of reactive oxygen species (ROS), we demonstrate that protein thiols can be modified by a reactive sulfenic acid to form disulfide adducts that undergo rapid cleavage in the presence of glutathione (GSH). As expected, 1,2-NQH(2)-SAc is rapidly hydrolyzed and partially oxidized to yield 1,2-NQ-SH, resulting in a redox cycling reaction that produces ROS through a chemical disproportionation reaction. The sulfenic acid forms of 1,2-NQ-SH and 1,2-NQH2-SH were detected by derivatization experiments with dimedone. 1,2-NQH(2)-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH(2) adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH(2)-SAc caused an extensive chemical modification of cellular proteins in both cases. Protein adduction by 1,2-NQ through a thio ether (C-S-C) bond slowly declined through a GSH-dependent S-transarylation reaction, whereas that originating from 1,2-NQH2-SAc through a disulfide (C-S-S-C) bond was rapidly restored to the free protein thiol in the cells. Under these conditions, 1,2-NQH(2)-SAc activated Nrf(2) and upregulated its target genes, which were enhanced by pretreatment with buthionine sulfoximine (BSO), to deplete cellular GSH. Pretreatment of catalase conjugated with poly(ethylene glycol) suppressed Nrf2 activation by 1,2-NQH(2)-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress.

DOI： 10.1021/tx500416y

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

Aims Dysregulation of autonomic nervous system activity can trigger ventricular arrhythmias and sudden death in patients with heart failure. N-type Ca2+ channels (NCCs) play an important role in sympathetic nervous system activation by regulating the calcium entry that triggers release of neurotransmitters from peripheral sympathetic nerve terminals. We have investigated the ability of NCC blockade to prevent lethal arrhythmias associated with heart failure.
Methods and results We compared the effects of cilnidipine, a dual N- and L-type Ca2+ channel blocker, with those of nitrendipine, a selective L-type Ca2+ channel blocker, in transgenic mice expressing a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). In this mouse model of dilated cardiomyopathy leading to sudden arrhythmic death, cardiac structure and function did not significantly differ among the control, cilnidipine, and nitrendipine groups. However, cilnidipine dramatically reduced arrhythmias in dnNRSF-Tg mice, significantly improving their survival rate and correcting the imbalance between cardiac sympathetic and parasympathetic nervous system activity. A beta-blocker, bisoprolol, showed similar effects in these mice. Genetic titration of NCCs, achieved by crossing dnNRSF-Tg mice with mice lacking CACNA1B, which encodes the alpha 1 subunit of NCCs, improved the survival rate. With restoration of cardiac autonomic balance, dnNRSF-Tg; CACNA1B(+/-) mice showed fewer malignant arrhythmias than dnNRSF-Tg; CACNA1B(+/+) mice.
Conclusions Both pharmacological blockade of NCCs and their genetic titration improved cardiac autonomic balance and prevented lethal arrhythmias in a mouse model of dilated cardiomyopathy and sudden arrhythmic death. Our findings suggest that NCC blockade is a potentially useful approach to preventing sudden death in patients with heart failure.

DOI： 10.1093/cvr/cvu185

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Structural and morphological changes of the cardiovascular systems (cardiovascular remodeling) are a major clinical outcome of cardiovascular diseases. Many lines of evidences have implied that transfiguration of reduction/oxidation (redox) homeostasis due to excess production of reactive oxygen species (ROS) and/or ROS-derived electrophilic metabolites (electrophiles) is the main cause of cardiovascular remodeling. Gasotransmitters, such as nitric oxide (NO) and endogenous electrophiles, are considered major bioactive species and have been extensively studied in the context of physiological and pathological cardiovascular events. We have recently found that hydrogen sulfide-related reactive species function as potent nucleophiles to eliminate electrophilic modification of signaling proteins induced by NO-derived electrophilic byproducts (e.g., 8-nitroguanosine 3',5'-cyclic monophosphate and nitro-oleic acid). In this review, we discuss the current understanding of redox control of cardiovascular pathophysiology by electrophiles and nucleophiles. We propose that modulation of electrophile-mediated post-translational modification of protein cysteine thiols may be a new therapeutic strategy of cardiovascular diseases. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

DOI： 10.1016/j.yjmcc.2014.02.003

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

Objective-Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI).
Approach and Results-We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca2+](i) in vitro and of vascular leakage in vivo were markedly attenuated by the Ca2+-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr(69) and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion.
Conclusions-ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.

DOI： 10.1161/ATVBAHA.113.001974

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

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

Authorship：Lead author   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

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

Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80 + macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.

DOI： 10.1038/ncomms2540

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

Reactive oxygen species (ROS) have been believed to be toxic substances that induce nonspecific damage in various biological molecules. ROS toxicology is now developing an emerging concept for physiological functions of ROS in the regulation of cell signal transductions. ROS signalling functions and their mechanisms are precisely regulated by several endogenous moderate electrophiles that are themselves generated from ROS during diverse physiological and pathophysiological cellular responses. The chemical biology of electrophiles is an emerging scientific area involving molecular mechanisms that conduct ROS cell signals through receptors to effector molecules at molecular, cellular and organism levels. The formation, signalling and metabolism of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) in cells are probably precisely regulated, and nonselective ROS reactions can be converted into stable, well-controlled electrophilic signal transduction via 8-nitro-cGMP. Modern redox biology is today advancing its frontier of basic research and clinical medicine, including infection, cancer biology, metabolic syndromes, ageing and even stem cell research. As one aspect of this advance, the 8-nitro-cGMP-mediated signalling that may be integrated into cells as a major redox signalling pathway may be a potential target in drug development and may lead to discovery of new therapeutic agents for various diseases. © The Authors 2012. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

DOI： 10.1093/jb/mvs145

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

Induction of cardiac fibrosis by β-blocker in G protein-independent and G protein-coupled receptor kinase 5/β-arrestin2-dependent Signaling pathways.
G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β(1)-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β(1)-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β(1)-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.

DOI： 10.1074/jbc.M112.357871

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

Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development. (C) 2012. Published by The Company of Biologists Ltd.

DOI： 10.1242/bio.20121370

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)  

Objective-The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)-induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition.
Methods and Results-Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II-induced contraction was largely dependent on C(a2+) influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)-dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II-induced Ca(2+) influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II-induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6.
Conclusion-PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II. (Arterioscler Thromb Vasc Biol. 2011;31:2278-2286.)

DOI： 10.1161/ATVBAHA.110.221010

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

Heart failure is a major cause of death in developed countries, and the development of an epoch-making cure is desired from the viewpoint for improving the quality of life and reducing the medical cost of the patient. The importance of neurohumoral factors, such as angiotensin (Ang) II and catecholamine, for the progression of heart failure has been supported by a variety of evidence. These agonists stimulate seven transmembrane-spanning receptors that are coupled to heterotrimeric GTP-binding proteins (G proteins). Using specific pharmacological tools to assess the involvement of G protein signaling pathways, we have revealed that á subunit of G _q (Gα _q) activates Ca ²⁺-dependent hypertrophic signaling through diacylglycerol-activated transient receptor potential canonical (TRPC) channels (TRPC3 and TRPC6: TRPC3/6). In contrast, activation of Gα ₁₂ family proteins in cardiomyocytes confers pressure overload-induced cardiac fibrosis via stimulation of purinergic P2Y ₆ receptors induced by extracellular nucleotides released from cardiomyocytes. In fact, direct or indirect inhibition of TRPC3/6 or P2Y ₆ receptors attenuates pressure overload-induced cardiac dysfunction. These findings will provide a new insight into the molecular mechanisms underlying pathogenesis of heart failure.

DOI： 10.1254/jphs.11R05CP

Language：English  

Regulation of Angiotensin II receptor signaling by cysteine modification of NF-kappa B
Angiotensin II (Ang II) is a major vasoactive peptide of the renin-angiotensin system. Ang Ills originally found as one of potent vasoconstrictors, but is now attracted attention as an essential mediator of many cardiovascular problems, including endothelial dysfunction, arrhythmia and structural remodeling of cardiovascular systems. Most of the known pathophysiological effects of Ang II are mediated through Ang type1 receptors (AT(1)Rs), and the up-regulation of AT(1)Rs is one of important causes by which Ang II can contribute to cardiovascular diseases. A growing body of evidence has suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in the regulation of AT(1)R signaling. In cardiac fibroblasts, stimulation with cytokines or bacterial toxins induces AT(1)R up-regulation through NADPH oxidase-dependent ROS production. In contrast, nitric oxide (NO) decreases AT(1)R density through cysteine modification (S-nitrosylation) of a transcriptional factor, nuclear factor kappa B (NF-kappa B). The difference between the effects of ROS and NO on AT(1)R expression may be caused by the difference between intracellular location of ROS signaling and that of NO signaling, as the agonist-induced S-nitrosylation of NF-kappa B requires a local interaction between NO synthase (NOS) and NF-kappa B in the perinuclear region. Thus, the spatial and temporal regulation of cysteine modification by ROS or RNS may underlie the resultant changes of AT(1)R signaling induced by agonist stimulation. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.

DOI： 10.1016/j.niox.2010.10.003

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca2+ signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (-/-)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca2+/calmodulin-dependent kinase II (CaMKII) were increased in MLP (-/-) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (-/-) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (-/-) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.04.124

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

Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT(1)R) density through NO-mediated S-nitrosylation of nuclear factor kappa B (NF-kappa B) in rat cardiac fibroblasts. Stimulation of purinergic P2Y(2) receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-kappa B in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. beta-Arrestins anchored the formation of p65/I kappa B alpha/beta-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-kappa B transcriptional activity and AT(1)R density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT(1)R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles.

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

We revealed that pre-treatment with docetaxel (DOC) 12 h before adriamycin (ADR) administration significantly reduced ADR-induced toxic death compared with the simultaneous dosing schedule that was commonly used in previous studies. We considered that pre-treatment with DOG relieves ADR-induced cardiotoxicity. In this study, we investigated the influence of DOG on the pharmacokinetics and pharmacodyamics of ADR in order to clarify the mechanism by which DOG pre-treatment relieves ADR-induced cardiotoxicity. When ADR and/or DOG was intravenously administered, the DOG pre-treatment (DOG-ADR) group showed significantly less toxic death than the ADR-alone group. We examined hepatopathy, nephropathy, leukopenia, and cardiotoxicity, all of which can cause toxic death. Of these toxicities, ADR-induced cardiotoxicity was significantly relieved in the DOG-ADR group. To elucidate the mechanism by which DOG pre-treatment relieved ADR-induced cardiotoxicity, lipid peroxidation as a proxy for the free radical level and the pharmacokinetics of ADR were measured. There was no difference in the pharmacokinetics of ADR between the ADR and DOG-ADR groups. On the other hand, the DOG-ADR group showed significantly inhibited lipid peroxidation in the heart compared with the ADR group. It was considered that DOG pre-administration inhibited ADR-induced free radicals and decreased cardiotoxicity.

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)  

Heterotrimeric G proteins are composed of α, β, and γ subunits. G proteins can be activated by a large number of cell-surface hepathelical receptors and can transduce signals from these receptors to various intracellular signaling molecules. When G protein-coupled receptors are bound by their cognate ligand, interaction with specific subtypes of G protein leads to dissociation of the α subunit of the heterotrimeric G protein from the βγ dimer, and both Gα-GTP and Gβγ are capable of initiating their own signal transduction pathways. G proteins are functionally divided into four groups based on the nature of α subunit into G _s, G_i, G_q, and G₁₂ families. The members of the G₁₂ subfamily are G₁₂ and G₁₃. Increasing evidence indicates that G_12/13 proteins play critical roles in various physiological functions. G₁₂ and G₁₃ regulate the small GTPase Rho through modulation of guanine nucleotide exchange factor (RhoGEF) activity to regulate various cellular responses, such as cytoskeletal changes and cell growth. Therefore, Rho activity can often represent a sensitive marker of G_12/13 activity. Here, we describe the Rho activation assay to monitor the activity of G_12/13 proteins.

DOI： 10.1007/978-1-61779-126-0_17

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

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from G(i) and Go through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1 beta among cytokines. Inhibition of IL-1 beta suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1 beta and AT1R through NF-kappa B, and a small GTP-binding protein, Rac, mediated PTX-induced NF-kappa B activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1 beta siRNA, suggesting that IL-1 beta-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of G(i)/G(o) by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of G(i)/G(o).

DOI： 10.1074/jbc.M109.076232

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Activation of Ca2+ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca2+ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-,diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca2+ influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca2+ spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca2+ influx itself or molecule(s) upstream of Ca2+ influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca2+ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.

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：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)  

Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

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)  

Cardiac hypertrophy is induced by various stresses such as hypertension and myocardial infarction. It is believed that hypertrophy is adaptive in the early phase but becomes maladaptive in the late phase. Cardiac hypertrophy develops heart failure when the heart is exposed persistently to the stresses. The increase in intracellular Ca²⁺ ([Ca²⁺]_i) plays an important role in the development of hypertrophy. It is generally thought that the increase in [Ca²⁺]_i for hypertrophy occurs via G_q-stimulated production of inositol-1,4,5-trisphosphate (IP ₃) and IP₃-mediated release of Ca²⁺ from intracellular store. However, several groups recently reported that canonical transient receptor potential (TRPC) channels are responsible for the increase in [Ca²⁺]_i. Among them, three TRPC subtypes (TRPC3/TRPC6/TRPC7) are activated by another G_q-mediated second messenger, diacylglycerol. Although several groups independently demonstrated that TRPC channels mediate receptor-stimulated and pressure overload-induced hypertrophy, there is discrepancy of which subtypes of TRPC channels predominantly mediate hypertrophy. However, there is consensus that TRPC-mediated Ca²⁺ influx is essential for hypertrophy. As TRPC channels participate in pathological hypertrophy, but not physiological contraction and the relaxation cycle, TPRC channels are a new target for the treatment of hypertrophy.

DOI： 10.1007/s00210-008-0321-8

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

Kelch-like ECH-associated protein 1 (Keap1), a BTB-Kelch substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex, regulates the induction of the phase 2 enzymes, such as glutathione S-transferase (GST), by repressing the transcription factor Nrf2. It is known that, in the human gastrointestinal tract, both GST A1 and P1 are constitutively expressed as the major GST isozymes. In the present study, using the Keap1-overexpressing derivatives of Caco-2 cells, human carcinoma cell line of colonic origin, by stable transfection of wild type Keap1, we investigated the molecular mechanism underlying the constitutive expression of these GST isozymes during differentiation. It was revealed that the overexpression of Keap1 completely repressed the constitutive expression of GST A1, but not GST P1. In Keap1-overexpressed cells, dome formation disappeared, and the formation of the intact actin cytoskeletal organization at cell-cell contact sites and the recruitment of E-cadherin and beta-catenin to adherens junctions were inhibited. The constitutive GST A1 expression in Caco-2 cells was repressed by disruption of E-cadherin-mediated cell-cell adhesion, suggesting the correlation between epithelial cell polarization and induction of the basal GST A1 expressions during Caco-2 differentiation. Keap1 overexpression indeed inhibited the activation of the small guanosine triphosphatase Rac1 on the formation of E-cadherin-mediated cell-cell adhesion. The transfection of V12Rac1, the constitutively active Rac1 mutant, into Keap1-overexpressed cells promoted the basal GST A1 expression, suggesting that Keap1 regulated the basal GST A1 expression during Caco-2 differentiation via Rac1 activation on the formation of E-cadherin-mediated cell-cell adhesion. The results of this study suggest the involvement of a novel Keap1-dependent signaling pathway for the induction of the constitutive GST A1 expression during epithelial cell differentiation.

DOI： 10.1021/bi800199z

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

Sustained elevation of [Ca2+](i) has been implicated in many cellular events. We previously reported that alpha subunits of G(12) family G proteins (G alpha(12/13)) participate in sustained Ca2+ influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that G alpha(12/13)-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca2+ influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active G alpha(12) or G alpha(13) increased the expression of TRPC6 proteins and basal Ca2+ influx activity. The treatment with ET-1 increased TRPC6 protein levels through G alpha(12/13), reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca2+](i) through up-regulated TRPC6. A G alpha(12/13)-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a G alpha(12/13)-reactive oxygen species-JNK pathway. The ET-1- induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of G alpha(12/13) in cardiac fibroblasts. First, G alpha(12/13) mediate ET-1- induced myofibroblast formation. Second, G alpha(12/13) mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts.

DOI： 10.1074/jbc.M611780200

Language：English  

Cognitive impairment and emotional disturbances in Alzheimer's disease (AD) result from the degeneration of synapses and neuronal death in the limbic system and associated regions of the cerebral cortex. An alteration in the proteolytic processing of the amyloid precursor protein (APP) results in increased production and accumulation of amyloid β-peptide (Aβ) in the brain. Aβ can render neurons vulnerable to excitotoxicity and apoptosis by disruption of cellular Ca²⁺ homeostasis and neurotoxic factors including reactive oxygen species (ROS), nitric oxide (NO), and cytokines. Many lines of evidence have suggested that transient receptor potential (TRP) channels consisting of six main subfamilies termed the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) are involved in Ca²⁺ homeostasis disruption. Thus, emerging evidence of the pathophysiological role of TRP channels has yielded promising candidates for molecular entities mediating Ca²⁺ homeostasis disruption in AD. In this review, we focus on the TRP channels in AD and highlight some TRP "suspects" for which a role in AD can be anticipated. An understanding of the involvement of TRP channels in AD may lead to the development of new target therapies.

DOI： 10.1016/j.bbadis.2007.03.006

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)  

Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the beta(2)-adrenergic receptor (beta(2)AR). However, the interaction of beta(2)AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of beta(2)AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis.

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)  

Sustained elevation in the intracellular Ca2+ concentration via Ca2+ influx, which is activated by a variety of mechanisms, plays a central regulatory role for cardiovascular functions. Recent molecular biological research has disclosed an unexpectedly diverse array of Ca2+-entry channel molecules involved in this Ca2+ influx. These include more than ten transient receptor potential (TRP) superfamily members such as TRPC1, TRPC3-6, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and polycystin (TRPP2). Most of them appear to be multimodally activated or modulated and show relevant features to both acute hemodynamic control and long-term remodeling of the cardiovascular system, and many of them have been found to respond not only to receptor stimulation but also to various forms of stimuli. There is good evidence to implicate TRPC1 in neointimal hyperplasia after vascular injury via store-depletion-operated Ca2+ entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca2+ mobilizations, being involved in vasoconstrictor and myogenic responses and pulmonary arterial proliferation and its associated disease (idiopathic pulmonary arterial hypertension). Considerable evidence has also been accumulated for unique involvement of TRPV1 in blood flow/pressure regulation via sensory vasoactive neuropeptide release. New lines of evidence suggest that TRPV2 may act as a Ca2+-overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg2+ entry channel, and TRPP2 as a Ca2+-entry channel requisite for vascular integrity. This review attempts to provide an overview of the current knowledge on TRP proteins and discuss their possible roles in cardiovascular functions and diseases.

DOI： 10.1161/01.RES.0000233356.10630.8a

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

Overexpression of constitutively active (CA)-G alpha(13) significantly increased the expression of interleukin (IL)-1 beta and IL-6 mRNAs and proteins in rat cardiac fibroblasts. IL-1 beta mRNA induction by CA-G alpha(13) was suppressed by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, but not by BAPTA-AM, an intracellular Ca2+ chelator. In contrast, IL-6 mRNA induction by CA-G alpha(13) was suppressed by BAPTA-AM but not by DPI. However, both IL-1 beta and IL-6 mRNA induction was suppressed by nuclear factor kappa B (NF-kappa B) inhibitors. The CA-G alpha(13)-induced NF-kappa B activation was suppressed by DPI and BAPTA-AM, but not C3 toxin and the Rho-kinase inhibitor Y27632. IL-6 mRNA induction by CA-G alpha(13) was suppressed by SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), an inhibitor of receptor-activated nonselective cation channels, and the expression of CA-G alpha(13) increased basal Ca2+ influx. These results suggest that G alpha(13) regulates IL-1 beta mRNA induction through the reactive oxygen species-NF-kappa B pathway, while it regulates IL-6 mRNA induction through the Ca2+-NF-kappa B pathway.

DOI： 10.1254/jphs.FP0051036

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

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

beta-Arrestin mediates desensitization and internalization of beta-adrenergic receptors (beta ARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, we have examined the role of beta-arrestin2 in the beta AR-mediated ERK signaling pathways. Isoproterenol stimulation equally activated cytoplasmic and nuclear ERK in COS-7 cells expressing beta(1)AR or beta(2)AR. However, the activity of nuclear ERK was enhanced by co-expression of beta-arrestin2 in beta(2)AR-but not beta(1)AR-expressing cells. Pertussis toxin treatment and blockade of G beta gamma action inhibited beta-arrestin2-enhanced nuclear activation of ERK, suggesting that beta-arrestin2 promotes nuclear ERK localization in a G beta gamma dependent mechanism upon receptor stimulation. beta(2)AR containing the carboxyl terminal region of beta(1)AR lost the beta-arrestin2-promoted nuclear translocation. As the carboxyl terminal region is important for beta-arrestin binding, these results demonstrate that recruitment of beta-arrestin2 to carboxyl terminal region of beta(2)AR is important for ERK localization to the nucleus. (c) 2005 Elsevier Inc. All rights reserved.

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)  

Cardiomyocytes express both beta(1)- and beta(2)-adrenergic receptors, and these receptors play a differential role in chronotropic and inotropic effects of the heart. Caveolae are known as an important regulator of G-protein-coupled receptor signaling. In the present report, we examined whether caveolae have a role in beta-adrenergic receptor-stimulated cAMP production and protein kinase A activation in neonatal myocytes. Isoproterenol-stimulated cAMP production was mediated by beta(1)- and beta(2)-subtypes, which depends on the receptor number of each subtype. However, protein kinase A activation was exclusively mediated by the beta(1)-subtype. Disruption of caveolae by methyl-beta-cyclodextrin treatment did not affect the relative contribution of subtypes to isoproterenol-stimulated cAMP production. beta(1)-Subtype-mediated protein kinase A activation was also not affected by the disruption of caveolae. These results suggest that beta(1)-adrenergic receptor-mediated protein kinase A activation is compartmentalized and independent of caveolae.

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

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH2-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the G alpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of G alpha(12/13), but not by an RGS domain specific for G alpha(q). Constitutively active G alpha(12)- or G alpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated G alpha(13), which was completely inhibited by the G alpha(12/13)-specific RGS domain. Furthermore, the G alpha(12/13)-specific but not the G alpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by G alpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that G alpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

DOI： 10.1074/jbc.M409710200

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

Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures(1,2). We previously mapped and narrowed a region associated with JME on chromosome 6p12-p11 (EJM1)(3-5). Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca2+ channel (Ca(v)2.3). EFHC1 and Ca(v)2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Cav2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca2+ currents that were reversed by the mutations associated with JME.

DOI： 10.1038/ng1393

Language：English  

Language：English  

Language：English  

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

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H2O2), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H2O2 to ECs markedly increased the BH4 level, but not its oxidized forms. The H2O2-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H2O2 induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H2O2-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H2O2. Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H2O2, and the H2O2-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H2O2 induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H2O2 appears to be one of the important signaling molecules to regulate the BH4-NOS system. (C) 2003 Elsevier Inc.

DOI： 10.1016/S0891-5849(03)00172-2

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

In neonatal cardiomyocytes, activation of the G(q)-coupled alpha(1)-adrenergic receptor (alpha(1)AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK). Here, we show that JNK activation is essential for alpha(1)AR-induced hypertrophy, in that alpha(1)AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate alpha(1)AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. alpha(1)AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Galpha(q), Galpha(12), and Galpha(13)- JNK activation was also inhibited by the Galpha(q/11)- or Galpha(12/13)-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters Gbetagamma. alpha(1)AR-induced hypertrophic responses were inhibited by Galpha(q/11)- and Galpha(12/13)-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Galpha(12) and Galpha(13) but not by Gaq. Our findings suggest that alpha(1)AR-induced hypertrophic responses are mediated in part by a Galpha(12/13)-Rho-JNK pathway, in part by a G(q/11)-JNK pathway that is Rho independent, and in part by a Gbetagamma pathway that is JNK independent.

DOI： 10.1161/01.RES.0000043282.39776.7C

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

In rat neonatal myocytes, a constitutively active Galpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the Gbetagamma-sequestering peptide betaARK-ct increases the phenylephrine-induced cardiac injury, indicating that Gbetagamma released from G(q) counteracts the Galpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by betaARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that Gbetagamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the Galpha(q)-induced cellular injury and apoptosis. (C) 2002 Elsevier Science (USA).

DOI： 10.1006/bbrc.2002.6553

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

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the a subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H2O2 is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe2+ suggesting that H2O2 is converted to more reactive species such as .OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with .OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in .OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Gai2 but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Gai2 by the mechanism similar to .OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.

DOI： 10.1074/jbc.M107392200

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

Capacitative Ca2+ entry (CCE) activated by release/depletion of Ca2+ from internal stores represents a major Ca2+ influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca2+ release-activated Ca2+ currents and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca2+ oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP3 receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca2+ signaling in B lymphocytes.

DOI： 10.1084/jem.20011758

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

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Call-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD(+)) to the MutT motif. Arachidonic acid and Call were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.

DOI： 10.1016/S1097-2765(01)00438-5

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Event date： 2012.3

Presentation type：Symposium, workshop panel (public)  

Venue：京都   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (invited, special)  

Venue：京都   Country：Japan  

Event date： 2011.11

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2011.11

Presentation type：Oral presentation (general)  

Venue：大阪   Country：Japan  

Event date： 2011.7

Presentation type：Oral presentation (general)  

Venue：北海道（小樽）   Country：Japan  

Event date： 2011.3

Presentation type：Oral presentation (general)  

Venue：横浜   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.6

Presentation type：Symposium, workshop panel (public)  

Venue：京都   Country：Japan  

Expression of Angiotensin (Ang) II type 1 receptor (AT1R) is one of major factors that determine Ang II-induced cardiovascular functions. We here demonstrated that AT1R-induced increase in intracellular Ca2+ concentration ([Ca2+]i increase) is inhibited by ATP treatment in rat cardiac fibroblasts. The number of AT1R was decreased by ATP. ATP increased nuclear factor of activated T cells (NFAT) activity through P2Y2 receptor. NFAT activation increased the expression of inducible nitric oxide synthase (iNOS), and the suppression of AT1R-induced [Ca2+]i increase by ATP was canceled by the inhibition of iNOS. Expression of AT1R is regulated by NF-κB activity and iNOS-induced NO inhibits NF-κB activity through S-nitrosylation of p65 subunit of NF-κB at Cys38. The suppression of AT1R-induced [Ca2+]i increase by ATP was canceled by overexpression of p65 (C38S) mutant. Furthermore, p65 was coprecipitated with iNOS and inhibition of p65-iNOS interaction canceled the S-nitrosylation of p65 induced by ATP stimulation. These results suggest that locally generated NO leads to S-nitrosylation of p65 at Cys38 via interaction of p65 with iNOS proteins, and S-nitrosylation of p65 underlies heterologous down-regulation of AT1R by purinergic P2Y2 receptor stimulation in rat cardiac fibroblasts.

Event date： 2010.5

Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2010.3

Presentation type：Symposium, workshop panel (public)  

Venue：岡山   Country：Japan  

Event date： 2010.3

Presentation type：Symposium, workshop panel (public)  

Venue：大阪   Country：Japan  

Event date： 2010.3

Presentation type：Symposium, workshop panel (public)  

Venue：大阪   Country：Japan  

Event date： 2010.3

Presentation type：Oral presentation (general)  

Venue：京都大学桂キャンパス   Country：Japan  

Event date： 2010.2

Presentation type：Symposium, workshop panel (public)  

Venue：Yeungnam University, Gyeongsan   Country：Korea, Republic of  

In excitable cardiomyocytes, changes in the frequency, shape, or amplitude of Ca2+ transients evoked by Ca2+ influx-induced Ca2+ release have been suggested to encode signals for induction of hypertrophy. A partial depolarization of plasma membrane by receptor stimulation is reported to increase the frequency of Ca2+ oscillations leading to activation of the nuclear factor of activated T cells (NFAT), a hypertrophic transcription factor that is predominantly regulated by calcineurin. Canonical transient receptor potential (TRPC) subfamily proteins were recently identified as a molecular candidate of receptor-activated cation channels. Among 7 TRPC homologues (C1-C7), diacylglycerol (DAG)-activated TRPC channels (C3, C6, C7) have been implicated in agonist-induced membrane depolarization in vascular systems. It has been thought that the sustained increase in intracellular Ca2+ concentration ([Ca2+]i) for hypertrophy occurs via Gq-stimulated production of inositol-1,4,5-trisphosphate (IP3) and IP3-mediated release of Ca2+ from intracellular store. However, we found that DAG-activated TRPC3 and TRPC6 channels are responsible for agonist-induced membrane depolarization in rat neonatal cardiomyocytes. As inhibition of either TRPC3 or TRPC6 completely suppressed agonist-induced NFAT activation and hypertrophic responses, TRPC3 and TRPC6 may form a heterotetramer channels. In addition, treatment with a TRPC3-selective blocker, pyrazole-3, attenuates pressure overload-induced cardiac hypertrophy and impairment of left ventricular function in mice. As TRPC channels participate in pathological hypertrophy but not physiological contraction and the relaxation cycle, these results suggest that DAG-activated TPRC channels are a new target for the treatment of heart failure.

Event date： 2009.11

Presentation type：Symposium, workshop panel (public)  

Venue：Huis Ten Bosch, Nagasaki   Country：Japan  

In excitable cardiomyocytes, changes in the frequency, shape, or amplitude of Ca2+ transients evoked by Ca2+ influx-induced Ca2+ release have been suggested to encode signals for induction of hypertrophy. A partial depolarization of plasma membrane by receptor stimulation is reported to increase the frequency of Ca2+ oscillations leading to activation of the nuclear factor of activated T cells (NFAT), a hypertrophic transcription factor that is predominantly regulated by calcineurin. Canonical transient receptor potential (TRPC) subfamily proteins were recently identified as a molecular candidate of receptor-activated cation channels. Among 7 TRPC homologues (C1-C7), diacylglycerol (DAG)-activated TRPC channels (C3, C6, C7) have been implicated in agonist-induced membrane depolarization in vascular systems. It has been thought that the sustained increase in intracellular Ca2+ concentration ([Ca2+]i) for hypertrophy occurs via Gq-stimulated production of inositol-1,4,5-trisphosphate (IP3) and IP3-mediated release of Ca2+ from intracellular store. However, we found that DAG-activated TRPC3 and TRPC6 channels are responsible for agonist-induced membrane depolarization in rat neonatal cardiomyocytes. As inhibition of either TRPC3 or TRPC6 completely suppressed agonist-induced NFAT activation and hypertrophic responses, TRPC3 and TRPC6 may form a heterotetramer channels. In addition, treatment with a TRPC3-selective blocker, pyrazole-3, attenuates pressure overload-induced cardiac hypertrophy and impairment of left ventricular function in mice. As TRPC channels participate in pathological hypertrophy but not physiological contraction and the relaxation cycle, these results suggest that DAG-activated TPRC channels are a new target for the treatment of heart failure.

Event date： 2009.9

Presentation type：Symposium, workshop panel (public)  

Venue：Joetsu Kokusai Ski Resort, Nagano   Country：Japan  

Event date： 2009.7

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

Venue：京都   Country：Japan  

Event date： 2009.7

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2009.3

Presentation type：Symposium, workshop panel (public)  

Venue：京都   Country：Japan  

Event date： 2009.3

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2008.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2024.7

Language：Japanese  

Venue：イギリス   Country：Japan  

Event date： 2024.4

Language：Japanese  

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：Symposium, workshop panel (public)  

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   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)  

Venue：神戸ポートアイランド   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

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

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：Fukuoka   Country：Japan  

Event date： 2023.10

Language：Japanese  

Country：Japan  

Event date： 2023.10

Language：Japanese  

Venue：沖縄   Country：Japan  

Event date： 2023.10

Language：Japanese  

Venue：岡崎   Country：Japan  

Event date： 2023.10

Language：Japanese  

Venue：岡崎   Country：Japan  

Event date： 2023.10

Language：Japanese  

Venue：岡崎   Country：Japan  

Event date： 2023.9

Language：Japanese  

Country：Japan  

Event date： 2023.9

Language：Japanese  

Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：秋田   Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：岡山   Country：Japan  

Event date： 2023.8 - 2023.9

Language：Japanese  

Venue：秋田   Country：Japan  

Event date： 2023.7

Language：Japanese  

Country：Japan  

Event date： 2023.7

Language：Japanese  

Country：Japan  

Event date： 2023.7

Language：Japanese  

Venue：山形   Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Venue：静岡   Country：Japan  

Event date： 2023.6

Language：Japanese  

Venue：静岡   Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：English  

Venue：仙台   Country：Japan  

Event date： 2023.6

Language：English  

Country：Korea, Republic of  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：English  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.6

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

Country：Japan  

Event date： 2023.6

Language：Japanese  

Country：Japan  

Event date： 2023.5

Language：Japanese  

Venue：岡崎   Country：Japan  

Event date： 2023.5

Language：Japanese  

Country：Japan  

Event date： 2023.5

Language：Japanese  

Country：Japan  

Event date： 2023.4

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.3

Language：Japanese  

Country：Japan  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.2

Language：Japanese  

Country：Japan  

Event date： 2023.1

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2023.1

Language：Japanese  

Country：Japan  

Event date： 2023.1

Language：Japanese  

Country：Japan  

Event date： 2022.12

Language：Japanese  

Country：Japan  

Event date： 2022.11

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

Venue：名古屋   Country：Japan  

Event date： 2022.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京   Country：Japan  

Event date： 2022.9

Language：Japanese  

Venue：淡路島   Country：Japan  

Event date： 2022.8

Language：Japanese  

Venue：静岡   Country：Japan  

Event date： 2022.7

Language：Japanese  

Venue：札幌   Country：Japan  

Event date： 2022.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌   Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Venue：熊本市   Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

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

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：English  

Venue：オンライン   Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：English  

Venue：オンライン   Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Country：Japan  

Event date： 2022.6

Language：Japanese  

Venue：オンライン開催   Country：Japan  

Event date： 2022.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：WEB   Country：Japan  

Event date： 2022.4

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：岡山   Country：Japan  

Event date： 2022.1 - 2023.1

Language：Japanese  

Country：Japan  

Event date： 2021.6

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

Venue：オンライン開催   Country：Japan  

Event date： 2021.3

Language：English  

Venue：オンライン開催   Country：Japan  

Event date： 2021.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪   Country：Japan  

Event date： 2021.1

Language：Japanese  

Country：Japan  

Event date： 2021.1

Language：Japanese  

Country：Japan  

Event date： 2021.1

Language：Japanese  

Country：Japan  

Event date： 2021.1

Language：Japanese  

Country：Japan  

Event date： 2021.1

Language：Japanese  

Country：Japan  

Event date： 2021.1

Language：Japanese  

Venue：大阪   Country：Japan  

Event date： 2021.1

Language：Japanese  

Venue：愛知   Country：Japan  

Event date： 2021.1

Language：Japanese  

Venue：香川   Country：Japan  

Event date： 2021.1

Language：Japanese  

Venue：東京   Country：Japan  

Event date： 2021.1

Language：English   Presentation type：Oral presentation (general)  

Venue：USA   Country：United States  

Event date： 2021.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：Seoul   Country：Korea, Republic of  

Event date： 2021.1

Language：English   Presentation type：Oral presentation (general)  

Venue：Seoul   Country：Korea, Republic of  

Event date： 2020.12

Language：English  

Venue：オンライン開催   Country：Japan  

Event date： 2020.12

Language：Japanese  

Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：オンライン   Country：Japan  

Event date： 2020.12

Language：Japanese  

Country：Japan  

Event date： 2020.11

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2020.11

Language：Japanese  

Venue：大阪   Country：Japan  

Event date： 2020.10

Language：Japanese  

Country：Japan  

Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2020.10

Language：Japanese  

Country：Japan  

Event date： 2020.9

Language：Japanese  

Country：Japan  

Event date： 2020.9 - 2020.4

Language：English  

Country：Japan  

Event date： 2020.9

Language：Japanese  

Country：Japan  

Event date： 2020.8

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2020.7

Language：Japanese  

Venue：仙台   Country：Japan  

Event date： 2020.7

Language：Japanese  

Venue：京都   Country：Japan  

Event date： 2020.7

Language：Japanese  

Venue：札幌   Country：Japan  

Event date： 2020.7

Language：Japanese  

Venue：徳島   Country：Japan  

Event date： 2020.7

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2020.7

Language：Japanese  

Venue：九重   Country：Japan