Faculty Profiles - TAKEFUJI Mikito

Papers - TAKEFUJI Mikito

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Urocortin2 measurement for heart failure assessment. Open Access

Inukai K, Miyashita K, Kotani K, Morimoto R, Kondo T, Hiraiwa H, Kazama S, Yoshida T, Yoshida S, Hattori T, Fukamachi K, Fukui N, Kato K, Yura Y, Ohashi K, Ouchi N, Murohara T, Okumura T, Takefuji M

Scientific reports Vol. 15 ( 1 ) page： 14381 2025.4

Language：English Publisher：Scientific Reports

Despite the efficacy of many therapies for heart failure, it remains a leading cause of morbidity and mortality worldwide, with many patients progressing to advanced stages of the condition. Since the standard treatment for heart failure includes small-molecule drugs targeting G protein-coupled receptors (GPCRs), GPCRs are still considered novel targets for the diagnosis and treatment of cardiovascular diseases. Corticotropin-releasing hormone receptor 2, a highly expressed GPCR in cardiomyocytes, and its ligand, urocortin2 (UCN2), have been reportedly associated with cardiovascular diseases; however, their clinical significance remains unclear. In this study, a UCN2 measurement assay was developed to measure blood UCN2 levels in patients with heart failure. The assay showed that blood UCN2 values indicated a negative relationship with cardiac ejection fraction in 52 patients with heart failure. Blood UCN2 levels were not correlated with brain natriuretic peptide, a standard marker of heart failure, and were higher in patients with cardiomyopathy than in those with heart failure, suggesting that measuring blood UCN2 levels may be a novel test for assessing the pathophysiology of heart failure.

DOI： 10.1038/s41598-025-99509-4

Pemafibrate ameliorates renal injury through induction of FGF21 and ketone body production in male mice. International journal Open Access

Kunihiko Takahara, Noriyuki Ouchi, Tomonobu Takikawa, Yuta Ozaki, Lixin Fang, Hiroshi Kawanishi, Minako Tatsumi, Yoshimitsu Yura, Katsuhiro Kato, Mikito Takefuji, Toyoaki Murohara, Koji Ohashi

Physiological reports Vol. 13 ( 3 ) page： e70135 2025.2

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

Chronic kidney disease is a life-threatening disease worldwide. PPARα is a crucial transcriptional regulator of lipid metabolism and inflammation. Here, we examine whether a novel selective PPARα modulator, pemafibrate modulates renal injury in a model of unilateral ureteral obstruction (UUO). Administration of pemafibrate to wild-type (WT) mice led to reduction of renal dysfunction and fibrosis after UUO with accompanying increases in plasma levels of fibroblast growth factor (FGF) 21 and ketone body β-hydroxybutyrate (BHB). Treatment of WT mice with FGF21 or BHB precursor resulted in attenuation of renal fibrotic and inflammatory responses after UUO. Treatment of proximal tubular cells with FGF21 or BHB reduced expression of epithelial-mesenchymal transition markers. These findings suggest that pemafibrate could ameliorate renal damage, at least in part, by its abilities to increase the production of FGF21 and BHB.

DOI： 10.14814/phy2.70135

ALPK2 prevents cardiac diastolic dysfunction in heart failure with preserved ejection fraction. International journal Open Access

Tatsuya Yoshida, Satoya Yoshida, Kohei Inukai, Katsuhiro Kato, Yoshimitsu Yura, Tomoki Hattori, Kentaro Taki, Atsushi Enomoto, Koji Ohashi, Takahiro Okumura, Noriyuki Ouchi, Haruya Kawase, Nina Wettschureck, Stefan Offermanns, Toyoaki Murohara, Mikito Takefuji

FASEB journal : official publication of the Federation of American Societies for Experimental Biology Vol. 38 ( 22 ) page： e70192 2024.11

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

Protein phosphorylation, controlled by protein kinases, is central to regulating various pathophysiological processes, including cardiac systolic function. The dysregulation of protein kinase activity plays a significant role in the pathogenesis of cardiac systolic dysfunction. While cardiac contraction mechanisms are well documented, the mechanisms underlying cardiac diastole remain elusive. This gap persists owing to the historical focus on systolic dysfunction in heart failure research. Recently, heart failure with preserved ejection fraction (HFpEF), an age-related disease characterized by cardiac diastolic dysfunction, has emerged as a major public health concern. However, its underlying mechanism remains unclear. In this study, we investigated cardiac protein kinases by analyzing the gene expression of 518 protein kinases in human tissues. We identified alpha-kinase 2 (ALPK2) as a novel cardiac-specific atypical kinase and generated tamoxifen-inducible, cardiomyocyte-specific Alpk2-knockout mice and Alpk2-overexpressing mice. Alpk2 deficiency did not affect cardiac systolic dysfunction in the myocardial infarction model or the pressure-overload-induced heart failure model. Notably, cardiomyocyte-specific Alpk2 deficiency exacerbated cardiac diastolic dysfunction induced by aging and in the HFpEF model. Conversely, Alpk2 overexpression increased the phosphorylation of tropomyosin 1, a major regulator that binds myosin to actin, and mitigated cardiac stiffness in HFpEF. This study provides novel evidence that ALPK2 represents a potential therapeutic target for cardiac diastolic dysfunction in HFpEF and age-related cardiac impairments.

DOI： 10.1096/fj.202402103R

Protein kinase N promotes cardiac fibrosis in heart failure by fibroblast-to-myofibroblast conversion. Reviewed International coauthorship International journal

Satoya Yoshida, Tatsuya Yoshida, Kohei Inukai, Katsuhiro Kato, Yoshimitsu Yura, Tomoki Hattori, Atsushi Enomoto, Koji Ohashi, Takahiro Okumura, Noriyuki Ouchi, Haruya Kawase, Nina Wettschureck, Stefan Offermanns, Toyoaki Murohara, Mikito Takefuji

Nature communications Vol. 15 ( 1 ) page： 7638 - 7638 2024.9

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

Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.

DOI： 10.1038/s41467-024-52068-0

Dynamic upregulation of retinoic acid signal in the early postnatal murine heart promotes cardiomyocyte cell cycle exit and maturation. International journal

Yusuke Fujikawa, Katsuhiro Kato, Kazumasa Unno, Shingo Narita, Yusuke Okuno, Yoshitaka Sato, Mikito Takefuji, Toyoaki Murohara

Scientific reports Vol. 14 ( 1 ) page： 20222 - 20222 2024.8

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

The adult mammalian heart has extremely limited cardiac regenerative capacity. Most cardiomyocytes live in a state of permanent cell-cycle arrest and are unable to re-enter the cycle. Cardiomyocytes switch from cell proliferation to a maturation state during neonatal development. Although several signaling pathways are involved in this transition, the molecular mechanisms by which these inputs coordinately regulate cardiomyocyte maturation are not fully understood. Retinoic acid (RA) plays a pivotal role in development, morphogenesis, and regeneration. Despite the importance of RA signaling in embryo heart development, little is known about its function in the early postnatal period. We found that mRNA expression of aldehyde dehydrogenase 1 family member A2 (Aldh1a2), which encodes the key enzyme for synthesizing all-trans retinoic acid (ATRA) and is an important regulator for RA signaling, was transiently upregulated in neonatal mouse ventricles. Single-cell transcriptome analysis and immunohistochemistry revealed that Aldh1a2 expression was enriched in cardiac fibroblasts during the early postnatal period. Administration of ATRA inhibited cardiomyocyte proliferation in cultured neonatal rat cardiomyocytes and human cardiomyocytes. RNA-seq analysis indicated that cell proliferation-related genes were downregulated in prenatal rat ventricular cardiomyocytes treated with ATRA, while cardiomyocyte maturation-related genes were upregulated. These findings suggest that RA signaling derived from cardiac fibroblasts is one of the key regulators of cardiomyocyte proliferation and maturation during neonatal heart development.

DOI： 10.1038/s41598-024-70918-1

Meflin/ISLR is a marker of adipose stem and progenitor cells in mice and humans that suppresses white adipose tissue remodeling and fibrosis.

Ishihara T, Kato K, Matsumoto K, Tanaka M, Hara A, Shiraki Y, Morisaki H, Urano Y, Ando R, Ito K, Mii S, Esaki N, Furuhashi K, Takefuji M, Suganami T, Murohara T, Enomoto A

Genes to cells : devoted to molecular & cellular mechanisms 2024.8

Language：English Publisher：Genes to Cells

Identifying specific markers of adipose stem and progenitor cells (ASPCs) in vivo is crucial for understanding the biology of white adipose tissues (WAT). PDGFRα-positive perivascular stromal cells represent the best candidates for ASPCs. This cell lineage differentiates into myofibroblasts that contribute to the impairment of WAT function. However, ASPC marker protein(s) that are functionally crucial for maintaining WAT homeostasis are unknown. We previously identified Meflin as a marker of mesenchymal stem cells (MSCs) in bone marrow and tissue-resident perivascular fibroblasts in various tissues. We also demonstrated that Meflin maintains the undifferentiated status of MSCs/fibroblasts. Here, we show that Meflin is expressed in WAT ASPCs. A lineage-tracing experiment showed that Meflin+ ASPCs proliferate in the WAT of obese mice induced by a high-fat diet (HFD), while some of them differentiate into myofibroblasts or mature adipocytes. Meflin knockout mice fed an HFD exhibited a significant fibrotic response as well as increases in adipocyte cell size and the number of crown-like structures in WAT, accompanied by impaired glucose tolerance. These data suggested that Meflin expressed by ASPCs may have a role in reducing disease progression associated with WAT dysfunction.

DOI： 10.1111/gtc.13154

Myonectin protects against skeletal muscle dysfunction in male mice through activation of AMPK/PGC1α pathway. International journal

Yuta Ozaki, Koji Ohashi, Naoya Otaka, Hiroshi Kawanishi, Tomonobu Takikawa, Lixin Fang, Kunihiko Takahara, Minako Tatsumi, Sohta Ishihama, Mikito Takefuji, Katsuhiro Kato, Yuuki Shimizu, Yasuko K Bando, Aiko Inoue, Masafumi Kuzuya, Shinji Miura, Toyoaki Murohara, Noriyuki Ouchi

Nature communications Vol. 14 ( 1 ) page： 4675 - 4675 2023.8

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

To maintain and restore skeletal muscle mass and function is essential for healthy aging. We have found that myonectin acts as a cardioprotective myokine. Here, we investigate the effect of myonectin on skeletal muscle atrophy in various male mouse models of muscle dysfunction. Disruption of myonectin exacerbates skeletal muscle atrophy in age-associated, sciatic denervation-induced or dexamethasone (DEX)-induced muscle atrophy models. Myonectin deficiency also contributes to exacerbated mitochondrial dysfunction and reduces expression of mitochondrial biogenesis-associated genes including PGC1α in denervated muscle. Myonectin supplementation attenuates denervation-induced muscle atrophy via activation of AMPK. Myonectin also reverses DEX-induced atrophy of cultured myotubes through the AMPK/PGC1α signaling. Furthermore, myonectin treatment suppresses muscle atrophy in senescence-accelerated mouse prone (SAMP) 8 mouse model of accelerated aging or mdx mouse model of Duchenne muscular dystrophy. These data indicate that myonectin can ameliorate skeletal muscle dysfunction through AMPK/PGC1α-dependent mechanisms, suggesting that myonectin could represent a therapeutic target of muscle atrophy.

DOI： 10.1038/s41467-023-40435-2

Adipolin protects against renal injury via PPARα-dependent reduction of inflammasome activation

Lixin Fang, Koji Ohashi, Satoko Hayakawa, Hayato Ogawa, Naoya Otaka, Hiroshi Kawanishi, Tomonobu Takikawa, Yuta Ozaki, Kunihiko Takahara, Minako Tatsumi, Mikito Takefuji, Yuuki Shimizu, Yasuko K. Bando, Yuya Fujishima, Norikazu Maeda, Iichiro Shimomura, Toyoaki Murohara, Noriyuki Ouchi

iScience Vol. 26 ( 5 ) page： 106591 - 106591 2023.5

How Should Weight Control and Caloric Intervention Be Implemented for Obese Heart Failure Patients?

Okumura T, Hiraiwa H, Takefuji M, Murohara T

Cardiology Vol. 148 ( 2 ) page： 103 - 105 2023

Benefits and Precautions in Using B-Type Natriuretic Peptide　― N-Terminal-Pro-B-Type Natriuretic Peptide Conversion Formula ―

Okumura Takahiro, Hiraiwa Hiroaki, Takefuji Mikito, Murohara Toyoaki

Circulation Journal Vol. 86 ( 12 ) page： 2019 - 2020 2022.11

Omentin attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-knockout mice. Reviewed International journal

Lixin Fang, Koji Ohashi, Naoya Otaka, Hayato Ogawa, Mizuho Hiramatsu-Ito, Hiroshi Kawanishi, Yasuko K Bando, Rei Shibata, Yuuki Shimizu, Katsuhiro Kato, Tomonobu Takikawa, Yuta Ozaki, Mikito Takefuji, Toyoaki Murohara, Noriyuki Ouchi

Cardiovascular research Vol. 118 ( 6 ) page： 1597 - 1610 2022.5

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

AIMS: Abdominal aortic aneurysm (AAA) is an increasing and life-threatening disease. Obesity contributes to an increased risk of AAA. Omentin is a circulating adipokine, which is downregulated in obese complications. Here, we examined whether omentin could modulate angiotensin (Ang) II-induced AAA formation in apolipoprotein E-knockout (apoE-KO) mice. METHODS AND RESULTS: apoE-KO mice were crossed with transgenic mice expressing the human omentin gene in fat tissue (OMT-Tg mice) to generate apoE-KO/OMT-Tg mice. apoE-KO/OMT-Tg and apoE-KO mice were subjected to continuous Ang II infusion by using osmotic mini pumps. apoE-KO/OMT-Tg mice exhibited a lower incidence of AAA formation and a reduced maximal diameter of AAA compared with apoE-KO mice. apoE-KO/OMT-Tg mice showed attenuated disruption of medial elastic fibres in response to Ang II compared with apoE-KO mice. apoE-KO/OMT-Tg mice also displayed reduced expression levels of matrix metalloproteinase (MMP) 9, MMP2, and pro-inflammatory genes in aortic walls compared with apoE-KO mice. Furthermore, systemic administration of omentin also attenuated AAA formation and disruption of medial elastic fibres in response to Ang II in apoE-KO mice. Treatment of human monocyte-derived macrophages with omentin protein attenuated expression of MMP9 and pro-inflammatory mediators, and MMP9 activation after stimulation with lipopolysaccharide. Treatment of human vascular smooth muscle cells (VSMCs) with omentin protein reduced expression and activation of MMP2 after stimulation with tumour necrosis factor α. Omentin treatment increased phosphorylation levels of Akt in human macrophages and VSMCs. The suppressive effects of omentin on MMP9 and MMP2 expression were reversed by inhibition of integrin-αVβ3/PI3-kinase/Akt signalling in macrophages and VSMCs, respectively. CONCLUSION: These data suggest that omentin acts as an adipokine that can attenuate Ang II-induced development of AAA through suppression of MMP9 and MMP2 expression and inflammatory response in the vascular wall.

DOI： 10.1093/cvr/cvab179

Serum autotaxin as a novel prognostic marker in patients with non-ischaemic dilated cardiomyopathy. Reviewed International journal

Takashi Araki, Takahiro Okumura, Hiroaki Hiraiwa, Takashi Mizutani, Yuki Kimura, Shingo Kazama, Naoki Shibata, Hideo Oishi, Tasuku Kuwayama, Toru Kondo, Ryota Morimoto, Mikito Takefuji, Toyoaki Murohara

ESC heart failure Vol. 9 ( 2 ) page： 1304 - 1313 2022.4

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

AIMS: Autotaxin (ATX) promotes myocardial inflammation, fibrosis, and the subsequent cardiac remodelling through lysophosphatidic acid production. However, the prognostic impact of serum ATX in non-ischaemic dilated cardiomyopathy (NIDCM) has not been clarified. We investigated the prognostic impact of serum ATX in patients with NIDCM. METHODS AND RESULTS: We enrolled 104 patients with NIDCM (49.8 ± 13.4 years, 76 men). We divided the patients into two groups using different cutoffs of median serum ATX levels for men and women: high-ATX group and low-ATX group. Cardiac events were defined as a composite of cardiac death and heart failure resulting in hospitalization. Median ATX level was 203.5 ng/mL for men and 257.0 ng/mL for women. Brain natriuretic peptide levels [224.0 (59.6-689.5) pg/mL vs. 96.5 (40.8-191.5) pg/mL, P = 0.010] were higher in the high-ATX group than low-ATX group, whereas high-sensitivity C-reactive protein and collagen volume fraction levels in endomyocardial biopsy samples were not significantly different between the two groups. Kaplan-Meier survival analysis revealed that the event-free survival rate was significantly lower in the high-ATX group than low-ATX group (log-rank; P = 0.007). Cox proportional hazard analysis revealed that high-ATX was an independent determinant of composite cardiac events. In both sexes, serum ATX levels did not correlate with high-sensitivity C-reactive protein levels and collagen volume fraction but had a weak correlation with brain natriuretic peptide levels (men; spearman's rank: 0.274, P = 0.017, women; spearman's rank: 0.378, P = 0.048). CONCLUSION: High serum ATX levels can be associated with increasing adverse clinical outcomes in patients with NIDCM. These results indicate serum ATX may be a novel biomarker or therapeutic target in NIDCM.

DOI： 10.1002/ehf2.13817

Factor Xa inhibitor, edoxaban ameliorates renal injury after subtotal nephrectomy by reducing epithelial-mesenchymal transition and inflammatory response. Reviewed International journal

Lixin Fang, Koji Ohashi, Hayato Ogawa, Naoya Otaka, Hiroshi Kawanishi, Tomonobu Takikawa, Yuta Ozaki, Kunihiko Takahara, Minako Tatsumi, Mikito Takefuji, Toyoaki Murohara, Noriyuki Ouchi

Physiological reports Vol. 10 ( 5 ) page： e15218 2022.3

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

Chronic kidney disease (CKD) is an increasing and life-threatening disease worldwide. Recent evidence indicates that blood coagulation factors promote renal dysfunction in CKD patients. Activated factor X (FXa) inhibitors are safe and first-line drugs for the prevention of thrombosis in patients with atrial fibrillation. Here, we investigated the therapeutic effects of edoxaban on CKD using the mouse 5/6 nephrectomy model. Eight-week-old wild-type mice were subjected to 5/6 nephrectomy surgery and randomly assigned to two groups, edoxaban or vehicle admixture diet. Edoxaban treatment led to reduction of urinary albumin excretion and plasma UN levels compared with vehicle group, which was accompanied with reduced glomerular cross-sectional area and cell number. Edoxaban treatment also attenuated fibrinogen positive area in the remnant kidneys after subtotal nephrectomy. Moreover, edoxaban treatment resulted in attenuated tubulointerstitial fibrosis after 5/6 nephrectomy, which was accompanied by reduced expression levels of epithelial-mesenchymal transition (EMT) markers, inflammatory mediators, and oxidative stress markers in the remnant kidneys. Treatment of cultured proximal tubular cells, HK-2 cells, with FXa protein led to increased expression levels of EMT markers, inflammatory mediators, and oxidative stress markers, which were abolished by pretreatment with edoxaban. Treatment of HK-2 cells with edoxaban attenuated FXa-stimulated phosphorylation levels of extracellular signal-regulated kinase (ERK) and NF-κB. Our findings indicate that edoxaban can improve renal injury after subtotal nephrectomy by reducing EMT and inflammatory response, suggesting that FXa inhibition could be a novel therapeutic target for CKD patients with atrial fibrillation.

DOI： 10.14814/phy2.15218

Neuron-derived neurotrophic factor protects against dexamethasone-induced skeletal muscle atrophy. Reviewed International journal

Yuta Ozaki, Koji Ohashi, Naoya Otaka, Hayato Ogawa, Hiroshi Kawanishi, Tomonobu Takikawa, Lixin Fang, Minako Tatsumi, Mikito Takefuji, Takashi Enomoto, Mohamed Darwish, Yoko Iijima, Takatoshi Iijima, Toyoaki Murohara, Noriyuki Ouchi

Biochemical and biophysical research communications Vol. 593 page： 5 - 12 2022.2

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

Skeletal muscle atrophy caused by various conditions including aging, nerve damage, and steroid administration, is a serious health problem worldwide. We recently reported that neuron-derived neurotrophic factor (NDNF) functions as a muscle-derived secreted factor, also known as myokine, which exerts protective actions on endothelial cell and cardiomyocyte function. Here, we investigated whether NDNF regulates skeletal muscle atrophy induced by steroid administration and sciatic denervation. NDNF-knockout (KO) mice and age-matched wild-type (WT) mice were subjected to continuous dexamethasone (DEX) treatment or sciatic denervation. NDNF-KO mice exhibited decreased gastrocnemius muscle weight and reduced cross sectional area of myocyte fiber after DEX treatment or sciatic denervation compared with WT mice. Administration of an adenoviral vector expressing NDNF (Ad-NDNF) or recombinant NDNF protein to gastrocnemius muscle of WT mice increased gastrocnemius muscle weight after DEX treatment. NDNF-KO mice showed increased expression of ubiquitin E3-ligases, including atrogin-1 and MuRF-1, in gastrocnemius muscle after DEX treatment, whereas Ad-NDNF reduced expression of atrogin-1 and MuRF-1 in gastrocnemius muscle of WT mice after DEX treatment. Pretreatment of cultured C2C12 myocytes with NDNF protein reversed reduced myotube diameter and increased expression of atrogin-1 and MuRF-1 after DEX stimulation. Treatment of C2C12 myocytes increased Akt phosphorylation. Pretreatment of C2C12 myotubes with the PI3-kinase/Akt inhibitor reversed NDNF-induced increase in myotube fiber diameter after DEX treatment. In conclusion, our findings indicated that NDNF prevents skeletal muscle atrophy in vivo and in vitro through reduction of ubiquitin E3-ligases expression, suggesting that NDNF could be a novel therapeutic target of muscle atrophy.

DOI： 10.1016/j.bbrc.2022.01.028

G-protein-mediated cellular signaling in heart failure

Takefuji Mikito

Shinzo Vol. 54 ( 2 ) page： 261 - 267 2022.2

Corticotropin releasing hormone receptor 2 antagonist, RQ-00490721, for the prevention of pressure overload-induced cardiac dysfunction. Reviewed International journal

Yu Mori, Ayako Tsuchihira, Tatsuya Yoshida, Satoya Yoshida, Akiyoshi Fujiuchi, Masashi Ohmi, Yumi Isogai, Teruhiro Sakaguchi, Shunsuke Eguchi, Takuma Tsuda, Katsuhiro Kato, Koji Ohashi, Noriyuki Ouchi, Hyi-Man Park, Toyoaki Murohara, Mikito Takefuji

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Vol. 146 page： 112566 - 112566 2022.2

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

BACKGROUND: G protein-coupled receptors (GPCRs) regulate the pathological and physiological functions of the heart. GPCR antagonists are widely used in the treatment of chronic heart failure. Despite therapeutic advances in the treatments for cardiovascular diseases, heart failure is a major clinical health problem, with significant mortality and morbidity. Corticotropin releasing hormone receptor 2 (CRHR2) is highly expressed in cardiomyocytes, and cardiomyocyte-specific deletion of the genes encoding CRHR2 suppresses pressure overload-induced cardiac dysfunction. This suggests that the negative modulation of CRHR2 may prevent the progression of heart failure. However, there are no systemic drugs against CRHR2. FINDINGS: We developed a novel, oral, small molecule antagonist of CRHR2, RQ-00490721, to investigate the inhibition of CRHR2 as a potential therapeutic approach for the treatment of heart failure. In vitro, RQ-00490721 decreased CRHR2 agonist-induced 3', 5'-cyclic adenosine monophosphate (cAMP) production. In vivo, RQ-00490721 showed sufficient oral absorption and better distribution to peripheral organs than to the central nervous system. Oral administration of RQ-00490721 inhibited the CRHR2 agonist-induced phosphorylation of cAMP-response element binding protein (CREB) in the heart, which regulates a transcription activator involved in heart failure. RQ-00490721 administration was not found to affect basal heart function in mice but protected them from pressure overload-induced cardiac dysfunction. INTERPRETATION: Our results suggest that RQ-00490721 is a promising agent for use in the treatment of chronic heart failure.

DOI： 10.1016/j.biopha.2021.112566

LPL/AQP7/GPD2 promotes glycerol metabolism under hypoxia and prevents cardiac dysfunction during ischemia. Reviewed International coauthorship International journal

Sohta Ishihama, Satoya Yoshida, Tatsuya Yoshida, Yu Mori, Noriyuki Ouchi, Shunsuke Eguchi, Teruhiro Sakaguchi, Takuma Tsuda, Katsuhiro Kato, Yuuki Shimizu, Koji Ohashi, Takahiro Okumura, Yasuko K Bando, Hiroaki Yagyu, Nina Wettschureck, Naoto Kubota, Stefan Offermanns, Takashi Kadowaki, Toyoaki Murohara, Mikito Takefuji

FASEB journal : official publication of the Federation of American Societies for Experimental Biology Vol. 35 ( 12 ) page： e22048 2021.12

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

In the heart, fatty acid is a major energy substrate to fuel contraction under aerobic conditions. Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply, making glucose the preferred substrate. However, the mechanism underlying the myocardial metabolic shift during ischemia remains unknown. Here, we show that lipoprotein lipase (LPL) expression in cardiomyocytes, a principal enzyme that converts triglycerides to free fatty acids and glycerol, increases during myocardial infarction (MI). Cardiomyocyte-specific LPL deficiency enhanced cardiac dysfunction and apoptosis following MI. Deficiency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes, increased the myocardial infarct size and apoptosis in response to ischemia. Ischemic conditions activated glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate adenosine triphosphate (ATP) synthesis from glycerol. Conversely, GPD2 deficiency exacerbated cardiac dysfunction after acute MI. Moreover, cardiomyocyte-specific LPL deficiency suppressed the effectiveness of peroxisome proliferator-activated receptor alpha (PPARα) agonist treatment for MI-induced cardiac dysfunction. These results suggest that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role in preventing myocardial ischemia-related damage.

DOI： 10.1096/fj.202100882R

Prognostic impact of transcardiac gradient of follistatin-like 1 reflecting hemodynamics in patients with dilated cardiomyopathy. Reviewed International journal

Hideo Oishi, Takahiro Okumura, Koji Ohashi, Yuki Kimura, Shingo Kazama, Naoki Shibata, Yoshihito Arao, Hiroo Kato, Tasuku Kuwayama, Shogo Yamaguchi, Minako Tatsumi, Toru Kondo, Hiroaki Hiraiwa, Ryota Morimoto, Mikito Takefuji, Noriyuki Ouchi, Toyoaki Murohara

Journal of cardiology Vol. 78 ( 6 ) page： 524 - 532 2021.12

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

BACKGROUND: Follistatin-like 1 (FSTL1) is a myocyte-secreted glycoprotein that could play a role in myocardial maintenance in response to harmful stimuli. We investigated the association between serum FSTL1 levels, especially focused on transcardiac gradient and the hemodynamics, to explore the prognostic impact of FSTL1 levels in patients with dilated cardiomyopathy (DCM). METHODS: Thirty-two ambulatory patients with DCM (23 men; mean age 59 years) were prospectively enrolled. Blood samples were simultaneously collected from the aortic root (Ao), coronary sinus (CS), as well as from the peripheral vein during cardiac catheterization in stable conditions. The transcardiac gradient of FSTL1 was calculated by the difference between serum FSTL1 levels of CS and Ao (FSTL1CS-Ao). Patients were divided into two groups based on the median of FSTL1CS-Ao: Low FSTL1CS-Ao group, <0 ng/mL; High FSTL1CS-Ao group, ≥0 ng/mL. Cardiac events were defined as a composite of cardiac deaths and hospitalizations for worsening heart failure. RESULTS: Mean left ventricular ejection fraction and median plasma B-type natriuretic peptide levels were 30.9% and 92.3 pg/mL, respectively. FSTL1CS-Ao was negatively correlated with pulmonary capillary wedge pressure (r = -0.400, p = 0.023). Kaplan-Meier survival analysis showed that event-free survival rate was significantly lower in the Low FSTL1CS-Ao group than in the High FSTL1CS-Ao group (p = 0.013). Cox regression analyses revealed that the transcardiac gradient of FSTL1 was an independent predictor for cardiac events. Receiver operating characteristic curve analysis showed that the cut-off value of FSTL1CS-Ao for the prediction of cardiac events was -4.09 ng/mL with sensitivity of 82% and specificity of 86% (area under the curve, 0.87). CONCLUSIONS: Fifty percent of patients had negative transcardiac gradient of FSTL1. Reduced transcardiac gradient of FSTL1 might be a novel prognostic predictor in DCM patients with impaired hemodynamics.

DOI： 10.1016/j.jjcc.2021.07.005

Serum autotaxin level predicts future cardiac events in patients with dilated cardiomyopathy Reviewed

Araki, T; Okumura, T; Mizutani, T; Kimura, Y; Kazama, S; Shibata, N; Oishi, H; Kuwayama, T; Hiraiwa, H; Kondo, T; Morimoto, R; Takefuji, M; Murohara, T

EUROPEAN HEART JOURNAL Vol. 42 page： 1730 - 1730 2021.10

Adverse Effect of Circadian Rhythm Disorder on Reparative Angiogenesis in Hind Limb Ischemia. International journal

Kazuhito Tsuzuki, Yuuki Shimizu, Junya Suzuki, Zhongyue Pu, Shukuro Yamaguchi, Yusuke Fujikawa, Katsuhiro Kato, Koji Ohashi, Mikito Takefuji, Yasuko K Bando, Noriyuki Ouchi, John W Calvert, Rei Shibata, Toyoaki Murohara

Journal of the American Heart Association Vol. 10 ( 16 ) page： e020896 2021.8

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

Background Circadian rhythm disorders, often seen in modern lifestyles, are a major social health concern. The aim of this study was to examine whether circadian rhythm disorders would influence angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia. Methods and Results A jet-lag model was established in C57BL/6J mice using a light-controlled isolation box. Control mice were kept at a light/dark 12:12 (12-hour light and 12-hour dark) condition. Concentrations of plasma vascular endothelial growth factor and circulating endothelial progenitor cells in control mice formed a circadian rhythm, which was diminished in the jet-lag model (P<0.05). The jet-lag condition deteriorated tissue capillary formation (P<0.001) and tissue blood perfusion recovery (P<0.01) in hind limb ischemia, which was associated with downregulation of vascular endothelial growth factor expression in local ischemic tissue and in the plasma. Although the expression of clock genes (ie, Clock, Bmal1, and Cry) in local tissues was upregulated after ischemic injury, the expression levels of cryptochrome (Cry) 1 and Cry2 were inhibited by the jet-lag condition. Next, Cry1 and Cry2 double-knockout mice were examined for blood perfusion recoveries and a reparative angiogenesis. Cry1 and Cry2 double-knockout mice revealed suppressed capillary density (P<0.001) and suppressed tissue blood perfusion recovery (P<0.05) in the hind limb ischemia model. Moreover, knockdown of CRY1/2 in human umbilical vein endothelial cells was accompanied by increased expression of WEE1 and decreased expression of HOXC5. This was associated with decreased proliferative capacity, migration ability, and tube formation ability of human umbilical vein endothelial cells, respectively, leading to impairment of angiogenesis. Conclusions Our data suggest that circadian rhythm disorder deteriorates reparative ischemia-induced angiogenesis and that maintenance of circadian rhythm plays an important role in angiogenesis.

DOI： 10.1161/JAHA.121.020896

Treatment with adipose-derived regenerative cells enhances ischemia-induced angiogenesis via exosomal microRNA delivery in mice. Reviewed

Tomohiro Kato, Katsuhiro Kato, Yuuki Shimizu, Mikito Takefuji, Toyoaki Murohara

Nagoya journal of medical science Vol. 83 ( 3 ) page： 465 - 476 2021.8

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

Adipose-derived regenerative cells (ADRCs), mesenchymal stem/progenitor cells from subcutaneous adipose tissue, have been shown to stimulate angiogenesis in hind limb ischemia, an effect attributed to paracrine action on endothelial cells (ECs) in mice. Despite promising therapeutic effects, the relevant molecules promoting neovascularization in this setting have not been fully elucidated. Extracellular vesicles, crucial mediators of intercellular communication, are recognized as a new therapeutic modality for regenerative medicine. Here, we found that GW4869, an exosome biogenesis inhibitor targeting neutral sphingomyelinase, impaired ADRCs-mediated angiogenesis and improvement of blood perfusion in a murine hind limb ischemia model. In addition, while the supernatant of ADRCs induced murine EC migration, this effect was attenuated by pre-treatment with GW4869. RNA analysis revealed that treatment of ADRCs with GW4869 reduced the expression of microRNA-21 (miR-21), miR-27b, miR-322, and let-7i in ADRCs-derived exosomes. Furthermore, the exosomes derived from GW4869-treated ADRCs induced the expression of the miR-21 targets Smad7 and Pten, and the miR-322 target Cul2, in ECs. These findings suggest that several miRNAs in ADRCs-derived exosomes contribute to angiogenesis and improvement of blood perfusion in a murine hind limb ischemia model.

DOI： 10.18999/nagjms.83.3.465

Meflin defines mesenchymal stem cells and/or their early progenitors with multilineage differentiation capacity. Reviewed International journal

Akitoshi Hara, Katsuhiro Kato, Toshikazu Ishihara, Hiroki Kobayashi, Naoya Asai, Shinji Mii, Yukihiro Shiraki, Yuki Miyai, Ryota Ando, Yasuyuki Mizutani, Tadashi Iida, Mikito Takefuji, Toyoaki Murohara, Masahide Takahashi, Atsushi Enomoto

Genes to cells : devoted to molecular & cellular mechanisms Vol. 26 ( 7 ) page： 495 - 512 2021.7

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

Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.

DOI： 10.1111/gtc.12855

Important Role of Concomitant Lymphangiogenesis for Reparative Angiogenesis in Hindlimb Ischemia. Reviewed International journal

Zhongyue Pu, Yuuki Shimizu, Kazuhito Tsuzuki, Junya Suzuki, Ryo Hayashida, Kazuhisa Kondo, Yusuke Fujikawa, Kazumasa Unno, Koji Ohashi, Mikito Takefuji, Yasuko K Bando, Noriyuki Ouchi, John W Calvert, Rei Shibata, Toyoaki Murohara

Arteriosclerosis, thrombosis, and vascular biology Vol. 41 ( 6 ) page： 2006 - 2018 2021.6

TOPICS 循環器内科学プロテインキナーゼNが関与する心不全の新規メカニズム

竹藤幹人

医学のあゆみ Vol. 276 ( 11 ) page： 1067 - 1068 2021.3

Adipolin/CTRP12 protects against pathological vascular remodeling through suppression of smooth muscle cell growth and macrophage inflammatory response. International journal

Ogawa H, Ohashi K, Ito M, Shibata R, Kanemura N, Yuasa D, Kambara T, Matsuo K, Hayakawa S, Hiramatsu-Ito M, Otaka N, Kawanishi H, Yamaguchi S, Enomoto T, Abe T, Kaneko M, Takefuji M, Murohara T, Ouchi N

Cardiovascular research Vol. 116 ( 1 ) page： 237 - 249 2020.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press (OUP)

<title>Abstract</title>
<sec>
<title>Aims</title>
Secreted factors produced by adipose tissue are involved in the pathogenesis of cardiovascular disease. We previously identified adipolin, also known as C1q/TNF-related protein 12, as an insulin-sensitizing adipokine. However, the role of adipolin in vascular disease remains unknown. Here, we investigated whether adipolin modulates pathological vascular remodelling.

</sec>
<sec>
<title>Methods and results</title>
Adipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to wire-induced injury of the femoral artery. APL-KO mice showed increased neointimal thickening after vascular injury compared with WT mice, which was accompanied by an enhanced inflammatory response and vascular cell proliferation in injured arteries. Adipolin deficiency also led to a reduction in transforming growth factor-β (TGF-β) 1 protein levels in injured arteries. Treatment of cultured macrophages with adipolin protein led to a reduction in lipopolysaccharide-stimulated expression of inflammatory mediators, including tumour necrosis factor (TNF)-α, interleukin (IL) 6, and monocyte chemotactic protein (MCP)-1. These effects were reversed by inhibition of TGF-β receptor II (TGF-βRII)/Smad2 signalling. Adipolin also reduced platelet-derived growth factor (PDGF)-BB-stimulated proliferation of vascular smooth muscle cells (VSMCs) through a TGF-βRII/Smad2-dependent pathway. Furthermore, adipolin treatment significantly increased TGF-β1 concentration in media from cultured VSMCs and macrophages.

</sec>
<sec>
<title>Conclusion</title>
These data indicate that adipolin protects against the development of pathological vascular remodelling by attenuating macrophage inflammatory responses and VSMC proliferation.

</sec>

DOI： 10.1093/cvr/cvz074

Other Link： http://academic.oup.com/cardiovascres/article-pdf/116/1/237/31555893/cvz074.pdf

Adipolin/C1q/Tnf-related protein 12 prevents adverse cardiac remodeling after myocardial infarction. Reviewed International journal

Tomonobu Takikawa, Koji Ohashi, Hayato Ogawa, Naoya Otaka, Hiroshi Kawanishi, Lixin Fang, Yuta Ozaki, Shunsuke Eguchi, Minako Tatsumi, Mikito Takefuji, Toyoaki Murohara, Noriyuki Ouchi

PloS one Vol. 15 ( 12 ) page： e0243483 2020

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

BACKGROUND: Myocardial infarction (MI) is a leading cause of death worldwide. We previously identified adipolin, also known as C1q/Tnf-related protein 12, as an anti-inflammatory adipokine with protective features against metabolic and vascular disorders. Here, we investigated the effect of adipolin on myocardial remodeling in a mouse model of MI. METHODS: Male adipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to the permanent ligation of the left anterior descending coronary artery to create MI. RESULTS: APL-KO mice exhibited increased ratios of heart weight/body weight and lung weight/body weight after MI compared with WT mice. APL-KO mice showed increased left ventricular diastolic diameter and decreased fractional shortening after MI compared with WT mice. APL-KO mice exhibited increased expression of pro-inflammatory mediators and enhanced cardiomyocyte apoptosis in the post-MI hearts compared with WT mice. Systemic administration of adenoviral vectors expressing adipolin to WT mice after MI surgery improved left ventricular contractile dysfunction and reduced cardiac expression of pro-inflammatory genes. Treatment of cultured cardiomyocytes with adipolin protein reduced lipopolysaccharide-induced expression of pro-inflammatory mediators and hypoxia-induced apoptosis. Treatment with adipolin protein increased Akt phosphorylation in cardiomyocytes. Inhibition of PI3 kinase/Akt signaling reversed the anti-inflammatory and anti-apoptotic effects of adipolin in cardiomyocytes. CONCLUSION: Our data indicate that adipolin ameliorates pathological remodeling of myocardium after MI, at least in part, by its ability to reduce myocardial inflammatory response and apoptosis.

DOI： 10.1371/journal.pone.0243483

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms Reviewed International journal

Kawanishi H, Ohashi K, Ogawa H, Otaka N, Takikawa T, Fang L, Ozaki Y, Takefuji M, Murohara T, Ouchi N

PLoS One. Vol. 15 ( 6 ) page： e0235362 - e0235362 2020

Language：English Publishing type：Research paper (scientific journal) Publisher：Public Library of Science (PLoS)

OBJECTIVE: Cardiovascular disease is a leading cause of death worldwide. Obesity-related metabolic disorders including dyslipidemia cause impaired collateralization under ischemic conditions, thereby resulting in exacerbated cardiovascular dysfunction. Pemafibrate is a novel selective PPARα modulator, which has been reported to improve atherogenic dyslipidemia, in particular, hypertriglyceridemia and low HDL-cholesterol. Here, we investigated whether pemafibrate modulates the revascularization process in a mouse model of hindlimb ischemia. METHODS AND RESULTS: Male wild-type (WT) mice were randomly assigned to two groups, normal diet or pemafibrate admixture diet from the ages of 6 weeks. After 4 weeks, mice were subjected to unilateral hindlimb surgery to remove the left femoral artery and vein. Pemafibrate treatment enhanced blood flow recovery and capillary formation in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of endothelial nitric oxide synthase (eNOS). Treatment of cultured endothelial cells with pemafibrate resulted in increased network formation and migratory activity, which were blocked by pretreatment with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Pemafibrate treatment also increased plasma levels of the PPARα-regulated gene, fibroblast growth factor (FGF) 21 in WT mice. Systemic administration of adenoviral vectors expressing FGF21 (Ad-FGF21) to WT mice enhanced blood flow recovery, capillary density and eNOS phosphorylation in ischemic limbs. Treatment of cultured endothelial cells with FGF21 protein led to increases in endothelial cell network formation and migration, which were canceled by pretreatment with L-NAME. Furthermore, administration of pemafibrate or Ad-FGF21 had no effects on blood flow in ischemic limbs in eNOS-deficient mice. CONCLUSION: These data suggest that pemafibrate can promote revascularization in response to ischemia, at least in part, through direct and FGF21-mediated modulation of endothelial cell function. Thus, pemafibrate could be a potentially beneficial drug for ischemic vascular disease.

DOI： 10.1371/journal.pone.0235362

Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction With Actin. Reviewed International coauthorship International journal

Teruhiro Sakaguchi, Mikito Takefuji, Nina Wettschureck, Tomonari Hamaguchi, Mutsuki Amano, Katsuhiro Kato, Takuma Tsuda, Shunsuke Eguchi, Sohta Ishihama, Yu Mori, Yoshimitsu Yura, Tatsuya Yoshida, Kazumasa Unno, Takahiro Okumura, Hideki Ishii, Yuuki Shimizu, Yasuko K Bando, Koji Ohashi, Noriyuki Ouchi, Atsushi Enomoto, Stefan Offermanns, Kozo Kaibuchi, Toyoaki Murohara

Circulation Vol. 140 ( 21 ) page： 1737 - 1752 2019.11

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

BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

DOI： 10.1161/CIRCULATIONAHA.119.041019

Cardiomyocytes capture stem cell-derived, anti-apoptotic microRNA-214 via clathrin-mediated endocytosis in acute myocardial infarction International journal

Eguchi Shunsuke, Takefuji Mikito (corresponding author, Sakaguchi Teruhiro, Ishihama Sohta, Mori Yu, Tsuda Takuma, Takikawa Tomonobu, Yoshida Tatsuya, Ohashi Koji, Shimizu Yuuki, Hayashida Ryo, Kondo Kazuhisa, Bando Yasuko K, Ouchi Noriyuki, Murohara Toyoaki

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 294 ( 31 ) page： 11665 - 11674 2019.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication that have the potential to improve cardiac function when used in cell-based therapy. However, the means by which cardiomyocytes respond to EVs remains unclear. Here, we sought to clarify the role of exosomes in improving cardiac function by investigating the effect of cardiomyocyte endocytosis of exosomes from mesenchymal stem cells on acute myocardial infarction (MI). Exposing cardiomyocytes to the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under hypoxia in vitro. In vivo, the injection of ADRCs into the heart simultaneous with coronary artery ligation decreased overall cardiac infarct area and prevented cardiac rupture after acute MI. Quantitative RT-PCR-based analysis of the expression of 35 known anti-apoptotic and secreted microRNAs (miRNAs) in ADRCs revealed that ADRCs express several of these miRNAs, among which miR-214 was the most abundant. Of note, miR-214 silencing in ADRCs significantly impaired the anti-apoptotic effects of the ADRC treatment on cardiomyocytes in vitro and in vivo. To examine cardiomyocyte endocytosis of exosomes, we cultured the cardiomyocytes with ADRC-derived exosomes labeled with the fluorescent dye PKH67 and found that hypoxic culture conditions increased the levels of the labeled exosomes in cardiomyocytes. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, significantly suppressed the ADRC-induced decrease of hypoxia-damaged cardiomyocytes and also decreased hypoxia-induced cardiomyocyte capture of both labeled EVs and extracellular miR-214 secreted from ADRCs. Our results indicate that clathrin-mediated endocytosis in cardiomyocytes plays a critical role in their uptake of circulating, exosome-associated miRNAs that inhibit apoptosis.

DOI： 10.1074/jbc.RA119.007537

Roles of the Mesenchymal Stromal/Stem Cell Marker Meflin in Cardiac Tissue Repair and the Development of Diastolic Dysfunction. International journal

Akitoshi Hara, Hiroki Kobayashi, Naoya Asai, Shigeyoshi Saito, Takahiro Higuchi, Katsuhiro Kato, Takahiro Okumura, Yasuko K Bando, Mikito Takefuji, Yasuyuki Mizutani, Yuki Miyai, Shoji Saito, Shoichi Maruyama, Keiko Maeda, Noriyuki Ouchi, Arata Nagasaka, Takaki Miyata, Shinji Mii, Noriyuki Kioka, Daniel L Worthley, Toyoaki Murohara, Masahide Takahashi, Atsushi Enomoto

Circulation research Vol. 125 ( 4 ) page： 414 - 430 2019.8

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

RATIONALE: Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. OBJECTIVE: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. METHODS AND RESULTS: We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-β, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-β and augments its intracellular signaling. CONCLUSIONS: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.

DOI： 10.1161/CIRCRESAHA.119.314806

Corticotropin-Releasing Hormone Family and Their Receptors in the Cardiovascular System

Takefuji Mikito, Murohara Toyoaki

Circulation Journal Vol. 83 ( 2 ) page： 261 - 266 2019.1

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

<p>The identification of corticotropin-releasing hormone (CRH) has led to the discovery of a growing family of ligands and receptors. CRH receptor 1 (CRHR1) and CRHR2 are mammalian G-protein coupled receptors (GPCRs) with high affinity for CRH and the CRH family of peptides. CRHR1 is predominantly expressed in the brain and plays a vital role in the hypothalamic-pituitary-adrenal (HPA) axis stress responses by secreting adrenal corticotropic hormone (ACTH). CRHR2 is predominantly expressed in the heart, and a CRHR2-specific ligand, urocortin 2 (UCN2), shows positive cardiac chronotropic and inotropic effects through 3´,5´-cyclic adenosine monophosphate (cAMP) signaling in response to CRHR2-mediated Gαs activation in mice and humans. Central administration of the CRH family of peptides increases mean arterial pressure through CRHR1 activation, whereas peripheral administration of the peptides decreases mean arterial pressure through CRHR2 activation. These observations have led to further investigations of CRHR2 as an important and unique GPCR in the physiological and pathological functioning of the cardiovascular (CV) system. Moreover, recent clinical trials demonstrate CRHR2 as a potentially therapeutic target in the treatment of heart failure. We present recent reviews of the role of CRHRs in basic CV physiology and in the pathophysiology of CV diseases.</p>

DOI： 10.1253/circj.CJ-18-0428

Monitoring of anti-Xa activity and factors related to bleeding events: A study in Japanese patients with nonvalvular atrial fibrillation receiving rivaroxaban International journal

Teruhiro Sakaguchi, Hiroyuki Osanai, Yosuke Murase, Hideki Ishii, Yoshihito Nakashima, Hiroshi Asano, Susumu Suzuki, Mikito Takefuji, Yasuya Inden, Kazuyoshi Sakai, Toyoaki Murohara, Masayoshi Ajioka

JOURNAL OF CARDIOLOGY Vol. 70 ( 3 ) page： 244 - 249 2017.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Background: Anti-Xa activity (AXA) in patients with nonvalvular atrial fibrillation (NVAF) and relationship to bleeding events remains unclear.
Methods: We evaluated AXA in 94 patients at both trough and peak rivaroxaban concentrations. Rivaroxaban dosage was determined according to creatinine clearance (CrCl): 10 and 15 mg once daily for patients with CrCl 15-49 and CrCl >= 50 mL/min, respectively. AXA value distribution and its association with bleeding events were examined in enrolled subjects.
Results: The mean peak AXA level was significantly higher than the mean trough level (1.98 +/- 0.81 vs. 0.16 +/- 0.15 IU/mL; p < 0.001). The peak AXA level significantly differed among patients with CrCl 15-29, 30-49, 50-79, and >= 80 mL/min (2.51 +/- 0.83, 1.72 +/- 0.76, 2.05 +/- 0.82, and 1.66 +/- 0.51 IU/mL, respectively; p = 0.004). Major and non-major clinically relevant bleeding events occurred in 22 patients (23.4% and 14.6% per year, respectively). The mean peak AXA level was significantly higher in patients who experienced bleeding events than in those who did not (2.40 +/- 0.70 vs. 1.84 +/- 0.80 IU/mL; p = 0.001). A Cox multivariate analysis showed that the peak AXA level was independently related to the incidence of major and non-major clinically relevant bleeding events (p = 0.012). Cumulative bleeding rates were significantly higher in patients with high peak AXA levels (p < 0.001).
Conclusion: Peak AXA level was an independent predictor for bleeding events in Japanese NVAF patients receiving rivaroxaban. (C) 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jjcc.2016.11.013

Dynamics of angiogenesis in ischemic areas of the infarcted heart International journal

Koichi Kobayashi, Kengo Maeda, Mikito Takefuji, Ryosuke Kikuchi, Yoshihiro Morishita, Masanori Hirashima, Toyoaki Murohara

SCIENTIFIC REPORTS Vol. 7 ( 1 ) page： 7156 - 7156 2017.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Cardiomyocytes are susceptible to apoptosis caused by hypoxia during the acute and subacute phases of myocardial infarction (MI). Angiogenesis can reduce MI-induced damage by mitigating hypoxia. It has been speculated that the ischemic border zone is a unique area rescued by angiogenic therapy. However, the mechanism and timing for new vessel formation in the mammalian heart following hypoxia are unclear. Identifying targets that benefit from angiogenesis treatment is indispensable for the development of revolutionary therapies. Here, we describe a novel circulatory system wherein new vessels develop from the endocardium of the left ventricle to perfuse the hypoxic area and salvage damaged cardiomyocytes at 3-14 days after MI by activating vascular endothelial growth factor signaling. Moreover, enhanced angiogenesis increased cardiomyocyte survival along the endocardium in the ischemic zone and suppressed ventricular remodeling in infarcted hearts. In contrast, cardiomyocytes in the border zone's hypoxic area underwent apoptosis within 12 h of MI, and the border area that was amenable to treatment disappeared. These data indicate that the non-perfused area along the endocardium is a site of active angiogenesis and a promising target for MI treatment.

DOI： 10.1038/s41598-017-07524-x

Corticotropin releasing hormone receptor 2 exacerbates chronic cardiac dysfunction Reviewed International coauthorship International journal

Takuma Tsuda, Mikito Takefuji, Nina Wettschureck, Kazuhiko Kotani, Ryota Morimoto, Takahiro Okumura, Harmandeep Kaur, Shunsuke Eguchi, Teruhiro Sakaguchi, Sohta Ishihama, Ryosuke Kikuchi, Kazumasa Unno, Kunihiro Matsushita, Shizukiyo Ishikawa, Stefan Offermanns, Toyoaki Murohara

JOURNAL OF EXPERIMENTAL MEDICINE Vol. 214 ( 7 ) page： 1877 - 1888 2017.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ROCKEFELLER UNIV PRESS

Heart failure occurs when the heart is unable to effectively pump blood and maintain tissue perfusion. Despite numerous therapeutic advancements over previous decades, the prognosis of patients with chronic heart failure remains poor, emphasizing the need to identify additional pathophysiological factors. Here, we show that corticotropin releasing hormone receptor 2 (Crhr2) is a G protein-coupled receptor highly expressed in cardiomyocytes and continuous infusion of the Crhr2 agonist, urocortin 2 (Ucn2), reduced left ventricular ejection fraction in mice. Moreover, plasma Ucn2 levels were 7.5-fold higher in patients with heart failure compared to those in healthy controls. Additionally, cardiomyocyte-specific deletion of Crhr2 protected mice from pressure overload-induced cardiac dysfunction. Mice treated with a Crhr2 antagonist lost maladaptive 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent signaling and did not develop heart failure in response to overload. Collectively, our results indicate that constitutive Crhr2 activation causes cardiac dysfunction and suggests that Crhr2 blockade is a promising therapeutic strategy for patients with chronic heart failure.

DOI： 10.1084/jem.20161924

Long-Term Pathological Follow-Up of Myocardium in a Carrier of Duchenne Muscular Dystrophy With Dilated Cardiomyopathy International journal

Toru Kondo, Takahiro Okumura, Mikito Takefuji, Hiroaki Hiraiwa, Yuki Sugiura, Naoki Watanabe, Soichiro Aoki, Takeo Ichii, Katsuhide Kitagawa, Naoaki Kano, Kenji Fukaya, Kenji Furusawa, Akinori Sawamura, Ryota Morimoto, Yasuko K. Bando, Genzou Takemura, Toyoaki Murohara

CIRCULATION-HEART FAILURE Vol. 10 ( 3 ) page： e003826 2017.3

Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart

Yura Yoshimitsu, Amano Mutsuki, Takefuji Mikito, Bando Tomohiro, Suzuki Kou, Kato Katsuhiro, Hamaguchi Tomonari, Hasanuzzaman Shohag Md., Takano Tetsuya, Funahashi Yasuhiro, Nakamuta Shinichi, Kuroda Keisuke, Nishioka Tomoki, Murohara Toyoaki, Kaibuchi Kozo

Cell Structure and Function Vol. 41 ( 2 ) page： 105 - 20 2016.8

Language：English Publisher：Japan Society for Cell Biology

<p>Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.</p>

DOI： 10.1247/csf.16011

Targeted Ablation of Periostin-Expressing Activated Fibroblasts Prevents Adverse Cardiac Remodeling in Mice Reviewed International journal

Harmandeep Kaur, Mikito Takefuji, C. Y. Ngai, Jorge Carvalho, Julia Bayer, Astrid Wietelmann, Ansgar Poetsch, Soraya Hoelper, Simon J. Conway, Helge Moellmann, Mario Looso, Christian Troidl, Stefan Offermanns, Nina Wettschureck

CIRCULATION RESEARCH Vol. 118 ( 12 ) page： 1906 - 17 2016.6

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Rationale: Activated cardiac fibroblasts (CF) are crucial players in the cardiac damage response; excess fibrosis, however, may result in myocardial stiffening and heart failure development. Inhibition of activated CF has been suggested as a therapeutic strategy in cardiac disease, but whether this truly improves cardiac function is unclear.
Objective: To study the effect of CF ablation on cardiac remodeling.
Methods and Results: We characterized subgroups of murine CF by single-cell expression analysis and identified periostin as the marker showing the highest correlation to an activated CF phenotype. We generated bacterial artificial chromosome-transgenic mice allowing tamoxifen-inducible Cre expression in periostin-positive cells as well as their diphtheria toxin-mediated ablation. In the healthy heart, periostin expression was restricted to valvular fibroblasts; ablation of this population did not affect cardiac function. After chronic angiotensin II exposure, ablation of activated CF resulted in significantly reduced cardiac fibrosis and improved cardiac function. After myocardial infarction, ablation of periostin-expressing CF resulted in reduced fibrosis without compromising scar stability, and cardiac function was significantly improved. Single-cell transcriptional analysis revealed reduced CF activation but increased expression of prohypertrophic factors in cardiac macrophages and cardiomyocytes, resulting in localized cardiomyocyte hypertrophy.
Conclusions: Modulation of the activated CF population is a promising approach to prevent adverse cardiac remodeling in response to angiotensin II and after myocardial infarction.

DOI： 10.1161/CIRCRESAHA.116.308643

Age-dependent blood pressure elevation is due to increased vascular smooth muscle tone mediated by G-protein signalling Reviewed International journal

Angela Wirth, Shengpeng Wang, Mikito Takefuji, Cong Tang, Till F. Althoff, Frank Schweda, Nina Wettschureck, Stefan Offermanns

CARDIOVASCULAR RESEARCH Vol. 109 ( 1 ) page： 131 - 140 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Aims Arterial hypertension is a major risk factor for cardiovascular diseases. The kidney and its natriuretic function are in the centre of the prevailing models to explain the pathogenesis of hypertension; however, the mechanisms underlying blood pressure elevation remain unclear in most patients. Development of hypertension is strongly correlated with age, and this blood pressure increase typically accelerates in the fourth decade of life. The cause of age-dependent blood pressure elevation is poorly understood. This study aims to understand the role of procontractile G-protein-mediated signalling pathways in vascular smooth muscle in age-dependent hypertension.
Methods and results Similar to humans at mid-life, we observed in 1-year-old mice elevated blood pressure levels without any evidence for increased vessel stiffness, impaired renal function, or endocrine abnormalities. Hypertensive aged mice showed signs of endothelial dysfunction and had an increased vascular formation of reactive oxygen species (ROS) and elevated endothelial ET-1 expression. Age-dependent hypertension could be normalized by ETA receptor blockade, smooth muscle-specific inactivation of the gene encoding the ETA receptor, as well as by acute disruption of downstream signalling via induction of smooth muscle-specific G alpha(12)/G alpha(13), G alpha(q)/G alpha(11), or LARG deficiency using tamoxifen-inducible smooth muscle-specific conditional mouse knock-out models. Induction of smooth muscle-specific ETA receptor deficiency normalized the blood pressure in aged mice despite the continuous presence of signs of endothelial dysfunction.
Conclusion Age-dependent blood pressure elevation is due to a highly reversible activation of procontractile signalling in vascular smooth muscle cells indicating that increased vascular tone can be a primary factor in the development of hypertension.

DOI： 10.1093/cvr/cvv249

Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart

Yura Yoshimitsu, Amano Mutsuki, Takefuji Mikito, Bando Tomohiro, Suzuki Kou, Kato Katsuhiro, Hamaguchi Tomonari, Shohag Md. Hasanuzzaman, Takano Tetsuya, Funahashi Yasuhiro, Nakamuta Shinichi, Kuroda Keisuke, Nishioka Tomoki, Murohara Toyoaki, Kaibuchi Kozo

CELL STRUCTURE AND FUNCTION Vol. 41 ( 2 ) page： 105 - 120 2016

Akt-dependent Girdin phosphorylation regulates repair processes after acute myocardial infarction.

Hayano S, Takefuji M, Maeda K, Noda T, Ichimiya H, Kobayashi K, Enomoto A, Asai N, Takahashi M, Murohara T

Journal of molecular and cellular cardiology Vol. 88 page： 55 - 63 2015.11

Language：English Publisher：Journal of Molecular and Cellular Cardiology

Myocardial infarction is a leading cause of death, and cardiac rupture following myocardial infarction leads to extremely poor prognostic feature. A large body of evidence suggests that Akt is involved in several cardiac diseases. We previously reported that Akt-mediated Girdin phosphorylation is essential for angiogenesis and neointima formation. The role of Girdin expression and phosphorylation in myocardial infarction, however, is not understood. Therefore, we employed Girdin-deficient mice and Girdin S1416A knock-in (GirdinSA/SA) mice, replacing the Akt phosphorylation site with alanine, to address this question. We found that Girdin was expressed and phosphorylated in cardiac fibroblasts in vitro and that its phosphorylation was crucial for the proliferation and migration of cardiac fibroblasts. In vivo, Girdin was localized in non-cardiomyocyte interstitial cells and phosphorylated in α-smooth muscle actin-positive cells, which are likely to be cardiac myofibroblasts. In an acute myocardial infarction model, GirdinSA/SA suppressed the accumulation and proliferation of cardiac myofibroblasts in the infarcted area. Furthermore, lower collagen deposition in GirdinSA/SA mice impaired cardiac repair and resulted in increased mortality attributed to cardiac rupture. These findings suggest an important role of Girdin phosphorylation at serine 1416 in cardiac repair after acute myocardial infarction and provide insights into the complex mechanism of cardiac rupture through the Akt/Girdin-mediated regulation of cardiac myofibroblasts.

DOI： 10.1016/j.yjmcc.2015.09.012

Alogliptin, a dipeptidyl peptidase-4 inhibitor, regulates the atrial arrhythmogenic substrate in rabbits. International journal

Toshihiko Yamamoto, Masayuki Shimano, Yasuya Inden, Mikito Takefuji, Satoshi Yanagisawa, Naoki Yoshida, Yukiomi Tsuji, Makoto Hirai, Toyoaki Murohara

Heart rhythm Vol. 12 ( 6 ) page： 1362 - 9 2015.6

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

BACKGROUND: Dipeptidyl peptidase-4 (DPP-4) inhibitors were recently reported to have cardioprotective effects via amelioration of ventricular function. However, the role of DPP-4 inhibition in atrial remodeling, especially of the arrhythmogenic substrate, remains unclear. OBJECTIVE: We investigated the effects of a DPP-4 inhibitor, alogliptin, on atrial fibrillation (AF) in a rabbit model of heart failure caused by ventricular tachypacing (VTP). METHODS: Rabbits subjected to VTP at 380 bpm for 1 or 3 weeks, with or without alogliptin treatment, were assessed using echocardiography, electrophysiology, histology, and immunoblotting and compared with nonpaced animals. RESULTS: VTP rabbits exhibited increased duration of atrial burst pacing-induced AF, whereas administration of alogliptin shortened this duration by 73%. The extent of atrial fibrosis after VTP was reduced by 39% in the alogliptin-treated group. VTP rabbits treated with alogliptin displayed a 1.6-fold increase in left atrial myocardial capillary density compared with nontreated rabbits. A 2-fold increase in endothelial nitric oxide synthase (eNOS) phosphorylation was observed in the left atrium of alogliptin-treated rabbits compared with nontreated rabbits. Moreover, a nitric oxide synthase inhibitor (N(ω)-nitro-l-arginine methyl ester) blocked the beneficial effects of alogliptin on AF duration, fibrosis, and capillary density. CONCLUSION: Alogliptin shortened the duration of AF caused by VTP-induced fibrotic atrial tissue by augmenting atrial angiogenesis and activating eNOS. Our findings suggest that DPP-4 inhibitors may be useful in the prevention of heart failure-induced AF.

DOI： 10.1016/j.hrthm.2015.03.010

Assessment of In-Stent Restenosis Using High-Definition Computed Tomography With a New Gemstone Detector

Tsuda Takuma, Ishii Hideki, Ichimiya Satoshi, Kanashiro Masaaki, Watanabe Jyunji, Takefuji Mikito, Aoyama Toru, Suzuki Susumu, Tanaka Akihito, Matsubara Tatsuaki, Murohara Toyoaki

Circulation Journal Vol. 79 ( 7 ) page： 1542 - 8 2015

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

<b><i>Background:</i></b>Until now, there have been few reports on the accuracy of in-stent restenosis (ISR) detection using high-definition computed tomography (HDCT). The purpose of this study was to assess ISR using HDCT with a new gemstone detector and to examine the diagnostic accuracy compared with invasive coronary angiography.<b><i>Methods and Results:</i></b>We evaluated 162 consecutive patients with 316 stents and the image quality (IQ) scores used to assess ISR, and analyzed whether stent strut thickness and diameter affected IQ score and assessability. In the 316 stents, 278 were diagnosed as assessable with HDCT (88.0%). IQ score for stent diameter ≥3 mm was significantly higher than that for stent diameter <3 mm, for stents with both thick struts ≥140 μm in thickness (mean IQ: 2.04±0.97 vs. 2.83±1.06, P<0.001) and thin struts <140 μm (mean IQ: 1.92±0.87 vs. 2.64±0.96, P=0.01). Assessability for stent diameter ≥3 mm was significantly higher than that for stent diameter <3 mm only for stents with thick struts (92.8% vs. 76.1%, P<0.001). Stent strut thickness, however, was not statistically significantly associated with either IQ score or assessability.<b><i>Conclusions:</i></b>In-stent lumens have high HDCT assessability, and HDCT is useful to evaluate thick-strut stents with diameter <3 mm. (<i>Circ J</i> 2015; <b>79:</b> 1542–1548)

DOI： 10.1253/circj.CJ-14-1344

Akt-dependent Girdin phosphorylation regulates repair processes after acute myocardial infarction. Reviewed

Hayano S, Takefuji M, Maeda K, Noda T, Ichimiya H, Kobayashi K, Enomoto A, Asai N, Takahashi M, Murohara T

J Mol Cell Cardiol. Vol. 88 page： 55-63 2015

RhoGEF-mediated vasoconstriction in hypertension.

Takefuji M, Yura Y, Kaibuchi K, Murohara T

Hypertension research : official journal of the Japanese Society of Hypertension Vol. 36 ( 11 ) page： 930 - 1 2013.11

RhoGEF12 controls cardiac remodeling by integrating G protein- and integrin-dependent signaling cascades.

Takefuji M, Krüger M, Sivaraj KK, Kaibuchi K, Offermanns S, Wettschureck N

The Journal of experimental medicine Vol. 210 ( 4 ) page： 665 - 73 2013.4

Language：English Publisher：Journal of Experimental Medicine

Structural cardiac remodeling, including hypertrophy and fibrosis, plays a crucial role in the pathogenesis of heart failure. In vitro studies suggested a role of the small GTPase RhoA in hypertrophic cardiomyocyte growth, but neither the molecular mechanisms leading to RhoA activation nor their relevance in vivo are known. We use here a mass spectrometric approach to identify Rho guanine nucleotide exchange factors (RhoGEFs) activated during cardiac pressure overload in vivo and show that RhoGEF12 is a central player during cardiac remodeling. We show that RhoGEF12 is required for stretch-induced RhoA activation and hypertrophic gene transcription in vitro and that its activation depends on integrin β1 and heterotrimeric G proteins of the G12/13 family. In vivo, cardiomyocyte-specific deletion of RhoGEF12 protects mice from overload-induced hypertrophy, fibrosis, and development of heart failure. Importantly, in mice with preexisting hypertrophy, induction of RhoGEF12 deficiency protects from cardiac decompensation, resulting in significantly increased longterm survival. Collectively, RhoGEF12 acts as an integrator of stretch-induced signaling cascades in cardiomyocytes and is an interesting new target for therapeutic intervention in patients with pressure overload-induced heart failure. © 2013 Takefuji et al.

DOI： 10.1084/jem.20122126

RhoGEF-mediated vasoconstriction in hypertension Reviewed

Takefuji M, Yura Y, Kaibuchi K, Murohara T

Hypertens Res. Vol. 36 ( 11 ) page： 930-931 2013

G13 controls angiogenesis through regulation of VEGFR-2 expression Reviewed International coauthorship

Sivaraj KK, Takefuji M, Schmidt I, Adams RH, Offermanns S, Wettchureck N

Dev Cell Vol. 25 ( 4 ) page： 427-434 2013

RhoGEF12 controls cardiac remodeling by intergrating G protein- and integrin-dependent signaling cascades. Reviewed

Takefuji M, Kruger M, Sivaraj KK, Kaibuchi K, Offermanns S, Wettshureck N

J Exp Med. Vol. 210 ( 4 ) page： 665-673 2013

G(13)-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure.

Takefuji M, Wirth A, Lukasova M, Takefuji S, Boettger T, Braun T, Althoff T, Offermanns S, Wettschureck N

Circulation Vol. 126 ( 16 ) page： 1972 - 82 2012.10

Procontractile G protein-mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling. Reviewed International coauthorship

Althoff TF, Juarez JA, Troidl K, Wirth A, Takefuji M, Wettschureck N,

J Exp Med. Vol. 209 ( 12 ) page： 2277-2290 2012

Mutation of ARHGAP9 in patients with coronary spastic angina.

Takefuji M, Asano H, Mori K, Amano M, Kato K, Watanabe T, Morita Y, Katsumi A, Itoh T, Takenawa T, Hirashiki A, Izawa H, Nagata K, Hirayama H, Takatsu F, Naoe T, Yokota M, Kaibuchi K

Journal of human genetics Vol. 55 ( 1 ) page： 42 - 9 2010.1

Language：English Publisher：Journal of Human Genetics

Coronary artery spasm has an important function in the etiology of variant angina and other acute coronary syndromes. Abnormal activation of Rho-family GTPases has been observed in cardiovascular disorders, but the function of genetic variability in Rho-family GTPases remains to be evaluated in cardiovascular disorders. We examined the genetic variability of Rho-family GTPases and their regulators in coronary artery spasm. We performed a comprehensive candidate gene analysis of 67 single nucleotide polymorphisms with amino-acid substitution in Rho-family GTPases and their regulators in 103 unrelated Japanese patients with acetylcholine-induced coronary artery spasm and 102 control Japanese subjects without acetylcholine-induced coronary artery spasm. We noted an association of the single nucleotide polymorphism of ARHGAP9 (rs11544238, Ala370Ser) with coronary artery spasm (odds ratio=2.67). We found that ARHGAP9 inactivated Rac as RacGAP and that the mRNA level of ARHGAP9 was strongly detected in hematopoietic cells. ARHGAP9 negatively regulated cell migration. The Ala370Ser polymorphism counteracted ARHGAP9-reduced cell migration, spreading and adhesion. The Ala370Ser polymorphism in the ARHGAP9 gene is associated with coronary artery spasm. These data suggest that the polymorphism of ARHGAP9 has a critical function in the infiltration of hematopoietic cells into the endothelium and inflammation leading to endothelial dysfunction. © 2010 The Japan Society of Human Genetics. All rights reserved.

DOI： 10.1038/jhg.2009.120

Rho-kinase Contributes to Sustained RhoA Activation through Phosphorylation of p190A RhoGAP Reviewed

Kazutaka Mori, Mutsuki Amano, Mikito Takefuji, Katsuhiro Kato, Yasuhiro Morita, Tomoki Nishioka, Yoshiharu Matsuura, Toyoaki Murohara, Kozo Kaibuchi

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 284 ( 8 ) page： 5067 - 76 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

RhoA is transiently activated by specific extracellular signals such as endothelin-1 (ET-1) in vascular smooth muscle cells. RhoGAP negatively regulates RhoA activity: thus, RhoA becomes the GDP-bound inactive form afterward. Sustained activation of RhoA is induced with high doses of the extracellular signals and is implicated in certain diseases such as vasospasms. However, it remains largely unknown how prolonged activation of RhoA is induced. Here we show that Rho-kinase, an effector of RhoA, phosphorylated p190A RhoGAP at Ser(1150) and attenuated p190A RhoGAP activity in COS7 cells. Binding of Rnd to p190A RhoGAP is thought to enhance its activation. Phosphorylation of p190A RhoGAP by Rho-kinase impaired Rnd binding. Stimulation of vascular smooth muscle cells with a high dose of ET-1 provoked sustained RhoA activation and p190A RhoGAP phosphorylation, both of which were prohibited by a Rho-kinase inhibitor. The phosphomimic mutation of p190A RhoGAP weakened Rnd binding and RhoGAP activities. Taken together, these results suggest that ET-1 induces Rho-kinase activation and subsequent phosphorylation of p190A RhoGAP, leading to prolonged RhoA activation.

DOI： 10.1074/jbc.M806853200

NOVEL MECHANISM OF SUSTAINED RHOA ACTIVATION IN VASCULAR SMOOTH MUSCLE; ITS POSSIBLE ROLE IN VASOSPASM

Katsuhiro Kato, Kazutaka Mori, Mutsuki Amano, Mikito Takefuji, Toyoaki Murohara, Kozo Kaibuchi

JOURNAL OF VASCULAR RESEARCH Vol. 46 page： 14 - 14 2009

39) 企業におけるAEDを用いた救命蘇生体験と救命救急体制づくり(第131回日本循環器学会東海地方会)

下方敬子, 近藤隆久, 大杉茂樹, 竹藤幹人, 梶口雅弘, 大林利博, 鈴木克昌

Circulation journal : official journal of the Japanese Circulation Society Vol. 72 ( 0 ) page： 1079 - 1079 2008.10

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation. International journal

Mikito Takefuji, Kazutaka Mori, Yasuhiro Morita, Nariko Arimura, Takashi Nishimura, Masanori Nakayama, Mikio Hoshino, Akihiro Iwamatsu, Toyoaki Murohara, Kozo Kaibuchi, Mutsuki Amano

Biochemical and biophysical research communications Vol. 355 ( 3 ) page： 788 - 94 2007.4

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

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.

DOI： 10.1016/j.bbrc.2007.02.028

Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase. International journal

Takako Kaneko, Akio Maeda, Mikito Takefuji, Hiroki Aoyama, Masanori Nakayama, Saeko Kawabata, Yoji Kawano, Akihiro Iwamatsu, Mutsuki Amano, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms Vol. 10 ( 10 ) page： 973 - 87 2005.10

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

After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated kinase (Rho-kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.

DOI： 10.1111/j.1365-2443.2005.00895.x

Potential role of Rap1 GTPase for cadherin turnover in MDCK cells

Jun Noritake, Kazumasa Sato, Masato Nakagawa, Nanae Izumi, Yuki Taya, Takashi Watanabe, Shuijie Wang, Mikito Takefuji, Masaki Fukata, Kozo Kaibuchi

CELL STRUCTURE AND FUNCTION Vol. 30 page： 35 - 35 2005.6

Rho-kinase and myosin II activities are required for cell type and environment specific migration. International journal

Masanori Nakayama, Mutsuki Amano, Akira Katsumi, Takako Kaneko, Saeko Kawabata, Mikito Takefuji, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms Vol. 10 ( 2 ) page： 107 - 17 2005.2

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

Cell migration is important in the development of atherosclerotic lesions. Macrophages and smooth muscle cells migrate into the subendothelial space of arteries, leading to plaque formation. Long-term inhibition of the activity of Rho-kinase induces a regression of atherosclerotic coronary lesions, probably by preventing migration of macrophages and smooth muscle cells. Previous reports concerning the effect of Rho-kinase inhibitors on cell migration are contradictory, however. We examined here the cell type specificity of Rho-kinase inhibitors and found that migration of endothelial cells, macrophages, and smooth muscle cells was inhibited by treatment with Rho-kinase inhibitors in a dose-dependent fashion in a three-dimensional migration assay, whereas that of fibroblasts and epithelial cells was not inhibited. Myosin II inhibitor prevented cell migration in a manner similar to Rho-kinase inhibitors. In contrast, in a two-dimensional migration assay, cell migration was not inhibited by Rho-kinase or myosin II inhibitors for any of the cell types examined. Taken together, these results indicate that Rho-kinase inhibitors suppress migration of specific cell types under specific conditions through the regulation of myosin II activity. Our findings suggest that Rho-kinase is the therapeutic target of atherosclerosis accompanied with invasion by leukocytes and smooth muscle cells.

DOI： 10.1111/j.1365-2443.2005.00823.x

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration.

Watanabe T, Wang S, Noritake J, Sato K, Fukata M, Takefuji M, Nakagawa M, Izumi N, Akiyama T, Kaibuchi K

Developmental cell Vol. 7 ( 6 ) page： 871 - 83 2004.12

Language：English Publisher：Developmental Cell

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration. Copyright © 2004 Cell Press.

DOI： 10.1016/j.devcel.2004.10.017

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration

Watanabe T, Wang SJ, Noritake J, Sato K, Fukata M, Takefuji M, Nakagawa M, Izumi N, Akiyama T, Kaibuchi K

DEVELOPMENTAL CELL Vol. 7 ( 6 ) page： 871 - 883 2004.12

Novel function of Rap1 GTPase in cadherin-mediated cell-cell adhesion

Noritake, J; Sato, K; Nakagawa, M; Izumi, N; Taya, Y; Watanabe, T; Wang, S; Takefuji, M; Fukata, M; Kaibuchi, K

MOLECULAR BIOLOGY OF THE CELL Vol. 15 page： 426A - 426A 2004.11

Role of Rap1 GTPase in regulation of cadherin-mediated cell-cell adhesion

Yuki Taya, Masato Nakagawa, Nanae Izumi, Jun Noritake, Takashi Watanabe, Shujie Wang, Kazumasa Sato, Mikito Takefuji, Kozo Kaibuchi

CELL STRUCTURE AND FUNCTION Vol. 29 page： 45 - 45 2004.5

Role of IQGAP1/APC complex in cell polarization and migration

Takashi Watanabe, Shujie Wang, Jun Noritake, Masaki Fukata, Masato Nakagawa, Mikito Takefuji, Nanae Izumi, Kazumasa Sato, Kozo Kaibuchi

CELL STRUCTURE AND FUNCTION Vol. 29 page： 33 - 33 2004.5

[Involvement of small GTPase Rho and Rho-kinase in the pathogenesis of hypertension and hypertensive target organ damage].

Nakayama M, Amano M, Kawabata S, Kaibuchi K, Takefuji M

Nihon rinsho. Japanese journal of clinical medicine Vol. 62 Suppl 3 page： 202 - 6 2004.3

Contribution of Rho/Rho-kinase pathway to cell migration in arteriosclerosis

Nakayama, M; Amano, M; Takefuji, M; Kaibuchi, K

JOURNAL OF PHARMACOLOGICAL SCIENCES Vol. 94 page： 94P - 94P 2004

Beneficial effect of cardiac resynchronization therapy on nocturnal disordered breathing in patients with chronic heart failure and intraventricular conduction delay

Y Yoshida, M Hirai, N Tsuboi, T Ito, M Takefuji, T Yamada, T Kondo, Y Inden, Y Murakami, H Hirayama, T Murohara, J Toyama

CIRCULATION Vol. 106 ( 19 ) page： 431 - 431 2002.11

[Intravenous vegetation of methicillin-resistant Staphylococcus aureus induced by central venous catheter in a patient with implantable cardioverter-defibrillator: a case report]. International journal

Kunihiro Matsushita, Naoya Tsuboi, Mamoru Nanasato, Mikito Takefuji, Natsuo Inoue, Taro Okada, Yukihiko Yoshida, Kazuo Hasegawa, Satoru Miwata, Haruo Hirayama, Teruo Ito, Wataru Kato, Kazuyoshi Tajima

Journal of cardiology Vol. 40 ( 1 ) page： 31 - 5 2002.7

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

A 54-year-old woman with an implantable cardioverter-defibrillator was admitted because of convulsion. Previously, she had suffered methicillin-resistant Staphylococcus aureus (MRSA) sepsis induced by a central venous catheter. Despite removal of the central venous catheter and administration of vancomycin, fever and increased C-reactive protein level persisted. MRSA had probably infected the implantable cardioverter-defibrillator system. Gallium scintigraphy showed accumulation at the right subclaviculan area, although local inflammatory signs were absent. Intravascular ultrasonography demonstrated intravenous vegetation at the bifurcation of the right subclavian vein and right internal jugular vein. Antibiotic therapy was not curative, so she underwent extirpation of the intravenous vegetation, which cured the systemic inflammation. Intravascular ultrasonography was useful to differentiate central venous catheter-related infection from implantable cardioverter defibrillator-related infection.

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