Faculty Profiles - TAKEFUJI Mikito

Papers - TAKEFUJI Mikito

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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

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